Inhibitors of matrix metallaproteinases

ABSTRACT

The present invention provides novel compounds of formulas I-IX, as described herein. Also provided are compositions of compounds of formulas I-IX, methods of making compounds of formulas I-IX, and methods of using compounds of formulas I-IX. The compounds of the invention can be used to inhibit matrix metalloproteinases, and are useful to treat conditions and diseases associated therewith.

RELATED APPLICATIONS

This application is a U.S. National Stage Filing under 35 U.S.C. 371from International Application Number PCT/US2006/019656, filed May 19,2006 and published in English as WO 2006/125208 on Nov. 23, 2006, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/743,467,filed Mar. 13, 2006, under 35 U.S.C. 119(e), which applications andpublication are incorporated herein by reference.

GOVERNMENT FUNDING

This invention was made with Government support under grant NCI-CA100475awarded by the National Institutes of Health. The United StatesGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

Specific interactions of cells within the extracellular matrix arecritical for the normal function of organisms. Alterations of theextracellular matrix are carried out by a family of zinc-dependentendopeptidases called matrix metalloproteinases (MMPs). The alterationsare carried out in various cellular processes such as organ development,ovulation, fetus implantation in the uterus, embryogenesis, woundhealing, and angiogenesis.

Twenty-six different MMPs are currently known. MMPs consist of fivemajor groups of enzymes: gelatinases, collagenases, stromelysins,membrane-type MMPs, and matrilysins. The activities of MMPs in normaltissue functions are strictly regulated by a series of complicatedzymogen activation processes and inhibition by protein tissue inhibitorsfor matrix metalloproteinases (TIMPs). Excessive MMP activity, when theregulation process fails, has been implicated in cancer growth, tumormetastasis, angiogenesis in tumors, arthritis and connective tissuediseases, cardiovascular disease, inflammation, autoimmune diseases,respiratory diseases, and neurological disorders.

Increased levels of activity for the human gelatinases MMP-2 and MMP-9have been implicated in several metabolic processes, for example,cancer, tumor metastasis, angiogenesis in tumors, arthritis andconnective tissue diseases, cardiovascular disease, inflammation,autoimmune diseases, respiratory diseases, and neurological disorders.Gelatinases are also of particular importance for both female ovulationand implantation of zygotes in the womb (for example, see U.S. Pat. No.6,703,415). As a result, selective inhibitors of MMPs are highly sought.

Several competitive inhibitors of MMPs are currently known. Theseinhibitors of MMPs take advantage of chelation to the active site zincfor inhibition of activity. Because of this general property, thesecompetitive inhibitors for MMPs are often toxic to the host, which hasbeen a major impediment in their clinical use.

Accordingly, there is a current need for new inhibitors of MMPs. Suchinhibitors would be useful to treat or prevent cancer, tumor metastasis,angiogenesis in tumors, contraception, arthritis and connective tissuediseases, cardiovascular disease, inflammation, autoimmune diseases,respiratory diseases, or neurological disorders. Also needed areinhibitors that exhibit selectivity for one or more specific MMPs. Suchinhibitors will preferably not include negative long-term side-effects.

SUMMARY OF THE INVENTION

The present invention relates to compounds of formulas I-IX,compositions that include compounds of formulas I-IX, methods of theirpreparation, and methods of their use. The pharmaceutical compositioncan include other therapeutic agents that are compatible with thecompound of the invention. The compounds can be used in medical therapy,for example to treat cancer, angiogenesis, cardiovascular disease,neurological disease, eye disease, inflammation, autoimmune disease, andfor regulating contraception, and other conditions that are affected bythe regulation of MMPs.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention may be best understood by referring to thefollowing detailed description and the accompanying drawings whichillustrate certain embodiments.

FIG. 1 is a schematic illustration wherein a coordinated thiirane moietyis predisposed to nucleophilic attack by the active site glutamate(Glu-404 in MMP-2) in MMP enzymes, a process that leads to covalentmodification of the enzyme and the attendant loss of activity.

FIG. 2 illustrates a stereoview of compound 1.3 bound to the active siteof MMP-2. A Connolly solvent-accessible surface is constructed in theactive site (shown in green), while the protein is rendered in purple.Compound 1.3, along with the active site Glu-404 and the threehistidines that are coordinated to the catalytic zinc are shown incapped-stick representation, colored according to atom types (yellow,red, blue, and white for S, O, N, and C, respectively). The zinc ion isshown as an orange sphere. The white arrow points to the S1′ pocket. Theside chain hydroxyl of Thr428 is expected to hydrogen bond (2.8 Å) tothe ester carbonyl of compound 1.3. The methyl group of compound 1.3 islocated near Leu399, Leu420, and Phe431, resulting in favorablehydrophobic interactions that would likely contribute to the overallbinding affinity.

FIG. 3 illustrates a schematic depiction of the mechanism of action ofinhibitor 2.1. (A) Coordination of the thiirane with the zinc ion is aprerequisite for the inhibition process. (B) Stereoview of thecomputational model for the non-covalent binding of inhibitor 2.1 in theactive site of MMP-2. The active site of the enzyme is depicted as aConnolly surface in green. The active site zinc ion is depicted as anorange sphere, with the three coordinating histidine residues depictedin capped sticks. Inhibitor 2.1 (in capped sticks and colored accordingto atom types) is shown coordinated to the active site zinc ion via thethiirane sulfur. The loop that constitutes the S₁′ subsite of the enzymeis drawn as a tube in purple, so the terminal phenyl group of theinhibitor is visible. The site of structure elaboration in arriving atmolecules 2.2-2.7 is indicated by the white arrow.

FIG. 4 illustrates slow-binding MMP inhibition by synthetic inhibitorsof Example 2. Progress curves were obtained by monitoring thefluorescence of the synthetic substrate MOCAcPLGLA₂pr(Dnp)AR-NH₂ (7 μM)in solutions of buffer R containing 0.5-1 nM of MMP-2 (A), MMP-9 (B) andMMP-14_(cat) (C) and inhibitor 2.3, as described under the ExperimentalProcedures section of Example 2. Inhibition of MMP-2 (0.5-1 nM) activityby compounds 2.5 (D) and 2.7 (E), under the same conditions. The linesrepresent nonlinear least-squares fits of the data to Equation 1, usingthe program Scientist. Insets, nonlinear least squares fits of theapparent rate constant k variation with inhibitor concentration toEquation 2, describing a one-step association mechanism. In FIG. 4,compounds 3, 5, and 7 refer to compounds 2.3, 2.5, and 2.7 of Example 2,respectively.

FIG. 5 illustrates equilibrium dialysis of MMP:inhibitor complexes.MMP-2 (A), MMP-9 (B) and MMP-14 (C) (10 nM each) were incubated in theabsence (▪) and presence of either compound 2.3 (

), 2.5 (

) and 2.7 (

) (1 mM each), in buffer R, for 3 hours, at room temperature. Theremaining MMP activity was monitored with MOCAcPLGLA₂pr(Dnp)AR-NH₂ (0hours). Part of the reaction mixtures was subjected to extensivedialysis against buffer R, containing no dimethyl sulfoxide, asdescribed under “Experimental Procedures” of Example 2, and theremaining solution was placed on a rotator. After 48 hours, theenzymatic activity in both the non-dialyzed (48 hours w/o dialysis) anddialyzed (48 hours w/dialysis) solutions was measured with theaforementioned fluorogenic substrate.

FIG. 6 illustrates that GM6001 (hydroxamate inhibitor), but notinhibitor 2.3, enhances MT1-MMP-dependent pro-MMP-2 activation by BS-C-1cells. BS-C-1 cells, co-infected to express MT1-MMP, as described underthe Experimental Procedures of Example 2, were incubated for 16 hourswith serum-free DMEM medium containing the indicated inhibitorconcentrations. After rinsing, the cells were incubated for 5 hours withserum-free DMEM supplemented with recombinant pro-MMP-2 (10 nM). (A) Themedia were collected and analyzed by gelatin zymography. L, I and Arefer to the latent, intermediate and active forms of MMP-2,respectively. (B) The cells were lysed and the lysates were subjected toimmunoblot analysis using the anti-MT1-MMP polyclonal antibody 815. The60- and 57-kDa forms represent pro- and active MT1-MMP, respectively.

FIG. 7 illustrates inhibition of HT1080 cell motility and invasion byinhibitor 2.3. A-B: Confluent cultures of HT1080 cells in 6-well plateswere treated with mitomycin C (25 μg/ml) in serum-free DMEM media for 30minutes. Scratch wounds were made on the monolayers and the woundedcultures were then incubated with serum-free DMEM supplemented withoutor with various amounts of inhibitor 2.3 (0-20 μM) for up to 20 hours.At each time period, the cultures were photographed (A) and the width ofthe scratch wound was measured as described under the ExperimentalProcedures section of Example 2. (C): HT1080 cells were seeded in 8-μmpore Transwell filters coated with Matrigel (50 μg/filter) in thepresence or absence of inhibitor 2.3 (0.1-10 μM). The number of cellsthat invaded to the lower side of the filter was counted in threerepresentative fields. Each value represents the mean±SE of fourindependent determinations. * P<0.05, and * P<0.001 when tested againstthe control using Tukey-Kramer Multiple Comparisons Test (P=0.004 byANOVA). In FIG. 7, inhibitor 3 refers to compound 2.3 of Example 2.

FIG. 8 illustrates specific compounds according to various embodimentsof the invention. These compounds have been prepared according to themethods described herein.

FIG. 9 illustrates certain specific and general compounds of theinvention, according to various embodiments.

DETAILED DESCRIPTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures, formulas, and Examples. While the invention will bedescribed in conjunction with the enumerated claims, it will beunderstood that they are not intended to limit the invention to thoseclaims. On the contrary, the invention is intended to cover allalternatives, modifications, and equivalents, which may be includedwithin the scope of the present invention as defined by the claims.

The present invention relates to compounds of formulas I-IX,compositions that include compounds of formulas I-IX, methods of theirpreparation, and methods of their use. When describing the compounds andmethods, the following terms have the following meanings, unlessotherwise indicated.

Definitions

References in the specification to “one embodiment”, “an embodiment”,“an example embodiment”, etc., indicate that the embodiment describedmay include a particular feature, structure, or characteristic, butevery embodiment may not necessarily include that particular feature,structure, or characteristic. Moreover, such phrases are not necessarilyreferring to the same embodiment. Further, when a particular feature,structure, or characteristic is described in connection with anembodiment, it is submitted that it is within the knowledge of oneskilled in the art to affect such feature, structure, or characteristicin connection with other embodiments whether or not explicitlydescribed.

“Substituted” is intended to indicate that one or more hydrogens on agroup indicated in the expression using “substituted” is replaced with aselection from the indicated group(s), provided that the indicatedatom's normal valency is not exceeded, and that the substitution resultsin a stable compound. Suitable indicated groups include, e.g., alkyl,alkenyl, alkylidenyl, alkenylidenyl, alkoxy, aryloxy, halo, haloalkyl,hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl,alkanoyl, alkoxycarbonyl, aroyl, acyloxy, aroyloxy, amino, imino,alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy,carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl,alkylsulfonyl, cyano, acetamido, acetoxy, acetyl, benzamido,benzenesulfinyl, benzenesulfonamido, benzenesulfonyl,benzenesulfonylamino, benzoyl, benzoylamino, benzoyloxy, benzyl,benzyloxy, benzyloxycarbonyl, benzylthio, carbamoyl, carbamate,isocyanato, sulfamoyl, sulfmamoyl, sulfmo, sulfo, sulfoamino, thiosulfo,NR^(x)R^(y) and/or COOR^(x), wherein each R^(x) and R^(y) areindependently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle,cycloalkyl or hydroxy. As would be readily understood by one skilled inthe art, when a substituent is keto (i.e., ═O) or thioxo (i.e., ═S), orthe like, then two hydrogen atoms on the substituted atom are replaced.

As used herein, the terms “optional” or “optionally” mean that thesubsequently described event or condition may but need not occur, andthat the description includes instances where the event or conditionoccurs and instances in which it does not. For example, “optionallysubstituted” means that the named substituent may be present but neednot be present, and the description includes situations where the namedsubstituent is included and situations where the named substituent isnot included.

Specific values described for radicals, substituents, and ranges, aswell as specific embodiments of the invention described herein, are forillustration only; they do not exclude other defined values or othervalues within defined ranges, as would be recognized by one skilled inthe art.

As used herein, the term “alkyl” refers to a branched, unbranched, orcyclic hydrocarbon having, for example, from 1 to 12 carbon atoms, andoften 1 to 6 carbon atoms. Examples include, but are not limited to,methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃), 1-propyl (n-Pr, n-propyl,—CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu,n-butyl, —CH₂CH₂CH₂CH₃), 2-methyl-1-propyl (1-Bu, i-butyl,—CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, —CH(CH₃)CH₂CH₃),2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl (n-pentyl,—CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂), and3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, hexyl, octyl, decyl, or dodecyl.The alkyl can be unsubstituted or substituted. The alkyl can also beoptionally partially or fully unsaturated. As such, the recitation of analkyl group includes both alkenyl and alkynyl groups. The alkyl can be amonovalent hydrocarbon radical, as described and exemplified above, orit can be a divalent hydrocarbon radical (i.e., alkylene).

The alkyl can optionally be substituted with one or more alkyl, alkenyl,alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy,hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl,alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro,trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo,alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, acetamido, acetoxy,acetyl, benzamido, benzenesulfinyl, benzenesulfonamido, benzenesulfonyl,benzenesulfonylamino, benzoyl, benzoylamino, benzoyloxy, benzyl,benzyloxy, benzyloxycarbonyl, benzylthio, carbamoyl, carbamate,isocyannato, sulfamoyl, sulfinamoyl, sulfino, sulfo, sulfoamino,thiosulfo, NR^(x)R^(y) and/or COOR^(x), wherein each R^(x) and R^(y) areindependently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle,cycloalkyl or hydroxy. The alkyl can optionally be interrupted with oneor more non-peroxide oxy (—O—), thio (—S—), imino (—N(H)—), methylenedioxy (—OCH₂O—), carbonyl (—C(═O)—), carboxy (—C(═O)O—), carbonyldioxy(—OC(═O)O—), carboxylato (—OC(═O)—), imine (C═NH), sulfinyl (SO) orsulfonyl (SO₂) groups.

The term “alkenyl” refers to a C₂-C₁₂ hydrocarbon containing normal,secondary, tertiary or cyclic carbon atoms with at least one site ofunsaturation, i.e. a carbon-carbon, sp2 double bond. Examples include,but are not limited to: ethylene or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂),cyclopentenyl (—C₅H₇), and 5-hexenyl (—CH₂ CH₂CH₂CH₂CH═CH₂). The alkenylcan be a movalent hydrocarbon radical, as described and exemplifiedabove, or it can be a divalent hydrocarbon radical (i.e., alkenylene).An alkenyl group can be substituted as described for alkyl groups above.

The term “alkynyl” refers to a monoradical branched or unbranchedhydrocarbon chain, having a point of complete unsaturation (i.e. acarbon-carbon, sp triple bond). In one embodiment, the alkynyl group canhave from 2 to 10 carbon atoms, or 2 to 6 carbon atoms. In anotherembodiment, the alkynyl group can have from 2 to 4 carbon atoms. Thisterm is exemplified by groups such as ethynyl, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 3-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl,1-octynyl, and the like. The alkynyl can be unsubstituted orsubstituted, as described above for alkyl groups.

The term “cycloalkyl” refers to cyclic alkyl groups of from 3 to 20carbon atoms having a single cyclic ring or multiple condensed rings.Such cycloalkyl groups include, by way of example, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, andthe like, or multiple ring structures such as adamantanyl, and the like.The cycloalkyl can optionally be partially unsaturated, therebyproviding a cycloalkenyl. The cycloalkyl group can be monovalent ordivalent, and can be optionally substituted as described above for alkylgroups.

The term “alkoxy” refers to the group alkyl-O—, where alkyl is asdefined herein. Preferred alkoxy groups include, e.g., methoxy, ethoxy,n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy,n-hexoxy, 1,2-dimethylbutoxy, and the like. Alkoxy groups can optionallybe substituted as described above for alkyl groups.

As used herein, “aryl” refers to an aromatic hydrocarbon group derivedfrom the removal of one hydrogen atom from a single carbon atom of aparent aromatic ring system. The radical can be at a saturated orunsaturated carbon atom of the parent ring system. The aryl group itselfcan have from 6 to 18 carbon atoms (excluding substituents). The arylgroup can have a single ring (e.g., phenyl) or multiple condensed(fused) rings, wherein at least one ring is aromatic (e.g., naphthyl,dihydrophenanthrenyl, fluorenyl, or anthryl). Typical aryl groupsinclude, but are not limited to, radicals derived from benzene,naphthalene, anthracene, biphenyl, and the like. The aryl can beunsubstituted or optionally substituted, as described above for alkylgroups.

The term “halo” refers to fluoro, chloro, bromo, and iodo. Similarly,the term “halogen” refers to fluorine, chlorine, bromine, and iodine.

The term “haloalkyl” refers to alkyl as defined herein substituted by 1or more halo groups as defined herein, which may be the same ordifferent. In one embodiment, the haloalkyl can be substituted with 1,2, 3, 4, or 5 halo groups. In another embodiment, the haloalkyl can bysubstituted with 1, 2, or 3 halo groups. The term haloalkyl also includeperfluoro-alkyl groups. Representative haloalkyl groups include, by wayof example, trifluoromethyl, 3-fluorododecyl, 12,12,12-trifluorododecyl,2-bromooctyl, 3-bromo-6-chloroheptyl, 1H,1H-perfluorooctyl, and thelike.

The term “heteroaryl” is defined herein as a monocyclic, bicyclic, ortricyclic ring system containing one, two, or three aromatic rings andcontaining at least one nitrogen, oxygen, or sulfur atom in an aromaticring, and which can be unsubstituted or substituted, for example, withone or more, and in particular one to three, substituents, as describedabove in the definition of “substituted”. Examples of heteroaryl groupsinclude, but are not limited to, 2H-pyrrolyl, 3H-indolyl,4H-quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl,β-carbolinyl, carbazolyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl,furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolisinyl,indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl,isoxazolyl, naphthyridinyl, oxazolyl, perimidinyl, phenanthridinyl,phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl,phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl,pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl,pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, tetrazolyl,and xanthenyl. In one embodiment the term “heteroaryl” denotes amonocyclic aromatic ring containing five or six ring atoms containingcarbon and 1, 2, 3, or 4 heteroatoms independently selected fromnon-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H, O,allyl, aryl, or (C₁-C₆)alkylaryl. In another embodiment heteroaryldenotes an ortho-fused bicyclic heterocycle of about eight to ten ringatoms derived therefrom, particularly a benz-derivative or one derivedby fusing a propylene, trimethylene, or tetramethylene diradicalthereto.

The term “heterocycle” refers to a saturated or partially unsaturatedring system, containing at least one heteroatom selected from the groupoxygen, nitrogen, and sulfur, and optionally substituted with one ormore groups as defined herein under the term “substituted”. Aheterocycle can be a monocyclic, bicyclic, or tricyclic group containingone or more heteroatoms. A heterocycle group also can contain an oxogroup (═O) attached to the ring. Non-limiting examples of heterocyclegroups include 1,3-dihydrobenzofuran, 1,3-dioxolane, 1,4-dioxane,1,4-dithiane, 2H-pyran, 2-pyrazoline, 4H-pyran, chromanyl,imidazolidinyl, imidazolinyl, indolinyl, isochromanyl, isoindolinyl,morpholine, piperazinyl, piperidine, piperidyl, pyrazolidine,pyrazolidinyl, pyrazolinyl, pyrrolidine, pyrroline, quinuclidine, andthiomorpholine. Other heterocycles include those described by Paquettein Principles of Modern Heterocyclic Chemistry (W. A. Benjamin, NewYork, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; and in TheChemistry of Heterocyclic Compounds. A Series of Monographs” (John Wiley& Sons, New York, 1950 to present), in particular Volumes 13, 14, 16,19, and 28; and J. Am. Chem. Soc. 1960, 82, 5566.

The term “alkanoyl” or “acyl” refers to —C(═O)R, wherein R is an alkylgroup as previously defined.

The term “aroyl” refers to —C(═O)Ar, wherein Ar is an aryl group aspreviously defined.

The term “alkoxycarbonyl” refers to —C(═O)OR, wherein R is an alkylgroup as previously defined.

The term “acyloxy” refers to —O—C(═O)R, wherein R is an alkyl group aspreviously defined. Examples of acyloxy groups include, but are notlimited to, acetoxy, propanoyloxy, butanoyloxy, and pentanoyloxy. Anyallyl group as defined above can be used to form an acyloxy group.

The term “amino” refers to —NH₂. The amino group can be optionallysubstituted as defined herein for the term “substituted”. The term“alkylamino” refers to —NR₂, wherein at least one R is alkyl and thesecond R is alkyl or hydrogen. The term “acylamino” refers toN(R)C(═O)R, wherein each R is independently hydrogen, alkyl, or aryl.

As to any of the above groups, which contain one or more substituents,it is understood, of course, that such groups do not contain anysubstitution or substitution patterns that are sterically impracticaland/or synthetically non-feasible. In addition, the compounds of thisinvention include all stereochemical isomers arising from thesubstitution of these compounds.

One diastereomer of a compound disclosed herein may display superiorproperties or activity compared with another. When required, separationof the racemic material can be achieved by HPLC using a chiral column orby a resolution using a resolving agent such as camphonic chloride asdescribed by Thomas J. Tucker, et al., J. Med. Chem. 1994, 37,2437-2444. A chiral compound may also be directly synthesized using achiral catalyst or a chiral ligand, e.g., as described by Mark A.Huffman, et al., J. Org. Chem. 1995, 60, 1590-1594.

Selected substituents within the compounds described herein are presentto a recursive degree. In this context, “recursive substituent” meansthat a substituent may recite another instance of itself. Because of therecursive nature of such substituents, theoretically, a large number maybe present in any given claim. One of ordinary skill in the art ofmedicinal chemistry and organic chemistry understands that the totalnumber of such substituents is reasonably limited by the desiredproperties of the compound intended. Such properties include, by ofexample and not limitation, physical properties such as molecularweight, solubility or log P, application properties such as activityagainst the intended target, and practical properties such as ease ofsynthesis.

Recursive substituents are an intended aspect of the invention. One ofordinary skill in the art of medicinal and organic chemistry understandsthe versatility of such substituents. To the degree that recursivesubstituents are present in an claim of the invention, the total numberwill be determined as set forth above.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent. Only stable compounds are contemplatedherein.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings or animals without excessive toxicity,irritation, allergic response, or other problems or complicationscommensurate with a reasonable benefit/risk ratio.

As used herein, “pharmaceutically acceptable salts” refer to compoundsdescribed herein, wherein the parent compound is modified by making acidor base salts thereof. Examples of pharmaceutically acceptable saltsinclude, but are not limited to, mineral or organic acid salts of basicresidues such as amines, and alkali or organic salts of acidic residuessuch as carboxylic acids; and the like. The pharmaceutically acceptablesalts include conventional non-toxic salts or quaternary ammonium saltsof the parent compound formed, for example, from inorganic or organicacids. For example, such conventional non-toxic salts include thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isethionic, and the like.

The pharmaceutically acceptable salts of the compounds described hereincan be synthesized from the parent compound, which contains a basic oracidic moiety, by conventional chemical methods. Generally, such saltscan be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 17th ed., Mack PublishingCompany, Easton, Pa., (1985), 1418, the disclosure of which isincorporated herein by reference.

As used herein, “treating” or “treat” includes preventing a pathologiccondition from occurring (e.g. prophylaxis); inhibiting the pathologiccondition or arresting its development; relieving a subject of thepathologic condition; and/or diminishing symptoms associated with thepathologic condition. “Treat,” “treating” or “treatment” includestreating, reversing, preventing, ameliorating, or inhibiting an injuryor disease-related condition or a symptom of an injury ordisease-related condition.

As used herein, “contacting” refers to the act of touching, makingcontact, or of bringing into immediate proximity.

The term “therapeutically effective amount” or “effective amount” isintended to include an amount of a compound described herein, or anamount of the combination of compounds described herein, e.g., to treator prevent a disease or disorder, or to treat the symptoms of a diseaseor disorder, typically in a host. The combination of compounds ispreferably a synergistic combination. Synergy, as described for exampleby Chou and Talalay (Adv. Enzyme Regul., 1984, 22, 27), occurs when theeffect of the compounds when administered in combination is greater thanthe additive effect of the compounds when administered alone as a singleagent. In general, a synergistic effect is most clearly demonstrated atsuboptimal concentrations of the compounds. Synergy can be in terms oflower cytotoxicity, increased activity, or some other beneficial effectof the combination compared with the individual components.

As used herein, a “therapeutic agent” is a compound that has biologicalactivity against any of tumor metastasis, angiogenesis in tumors,cancer, arthritis and connective tissue diseases, cardiovasculardisease, inflammation, autoimmune diseases, respiratory diseases, orneurological disorders. The therapeutic agent can be administered to apatient with a compound of formulas I-IX without losing its therapeuticactivity. Suitable therapeutic agents include, e.g., anti-inflammatoryagents, antibiotics, anti-viral agents, anticoagulants, α-adrenergicagonists, β-adrenergic agonists, analgesics, antineoplasts, adjuncts,androgen inhibitors, antibiotic derivatives, antiestrogens,antimetabolites, cytotoxic agents, hormones, immunomodulators, nitrogenmustard derivatives and steroids. Other therapeutic agents that can beused in conjunction with the compounds of the invention are disclosed inthe Physicians' Desk Reference, 59th Ed.; Thompson PDR: Montvale, N.J.(2005).

A “subject” can be a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, humans, farm animals,sport animals, and companion animals.

The term “protecting group” refers to any group which, when bound to ahydroxyl, nitrogen, or other heteroatom prevents undesired reactionsfrom occurring at this group and which can be removed by conventionalchemical or enzymatic steps to reestablish the hydroxyl group. Theparticular removable blocking group employed is not critical andpreferred removable hydroxyl blocking groups include conventionalsubstituents such as, for example, allyl, benzyl, acetyl, chloroacetyl,thiobenzyl, benzylidine, phenacyl, methyl methoxy, silyl ethers (e.g.,trimethylsilyl (TMS), t-butyl-diphenylsilyl (TBDPS), ort-butyldimethylsilyl (TBS)) and any other group that can be introducedchemically onto a hydroxyl functionality and later selectively removedeither by chemical or enzymatic methods in mild conditions compatiblewith the nature of the product.

A large number of protecting groups and corresponding chemical cleavagereactions are described in Protective Groups in Organic Synthesis,Theodora W. Greene (John Wiley & Sons, Inc., New York, 1991, ISBN0-471-62301-6) (“Greene”, which is incorporated herein by reference inits entirety). Included therein are nitrogen protecting groups, forexample, amide-forming groups. In particular, see Chapter 1, ProtectingGroups: An Overview, pages 1-20, Chapter 2, Hydroxyl Protecting Groups,pages 21-94, Chapter 4, Carboxyl Protecting Groups, pages 118-154, andChapter 5, Carbonyl Protecting Groups, pages 155-184. See alsoKocienski, Philip J.; Protecting Groups (Georg Thieme Verlag Stuttgart,New York, 1994), which is incorporated herein by reference in itsentirety. Some specific protecting groups that can be employed inconjunction with the methods of the invention are discussed below.

Typical nitrogen protecting groups described in Greene (pages 14-118)include benzyl ethers, silyl ethers, esters including sulfonic acidesters, carbonates, sulfates, and sulfonates. For example:

-   -   substituted methyl ethers;    -   substituted ethyl ethers;    -   p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl;    -   substituted benzyl ethers (p-methoxybenzyl, 3,4-dimethoxybenzyl,        o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl,        p-cyanobenzyl, p-phenylbenzyl, 2- and 4-picolyl, diphenylmethyl,        5-dibenzosuberyl, triphenylmethyl,        p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,        tri(p-methoxyphenyl)methyl, 1,3-benzodithiolan-2-yl,        benzisothiazolyl S,S-dioxido);    -   silyl ethers (silyloxy groups) (trimethylsilyl, triethylsilyl,        triisopropylsilyl, dimethylisopropylsilyl,        diethylisopropylsilyl, dimethylthexylsilyl,        t-butyldimethylsilyl, t-butyldiphenylsilyl, tribenzylsilyl,        tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl,        t-butylmethoxyphenylsilyl);    -   esters (formate, benzoylformate, acetate, choroacetate,        dichloroacetate, trichloroacetate, trifluoroacetate,        methoxyacetate, triphenylmethoxyacetate, phenoxyacetate,        p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate        (levulinate), pivaloate, adamantoate, crotonate,        4-methoxycrotonate, benzoate, p-phenylbenzoate,        2,4,6-trimethylbenzoate (mesitoate));    -   carbonates (methyl, 9-fluorenylmethyl, ethyl,        2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl,        2-(phenylsulfonyl)ethyl, 2-(triphenylphosphonio)ethyl, isobutyl,        vinyl, allyl, p-nitrophenyl, benzyl, p-methoxybenzyl,        3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, S-benzyl        thiocarbonate, 4-ethoxy-1-naphthyl, methyl dithiocarbonate);    -   groups with assisted cleavage (2-iodobenzoate, 4-azidobutyrate,        4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,        2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl carbonate,        4-(methylthiomethoxy)butyrate,    -   miscellaneous esters (2,6-dichloro-4-methylphenoxyacetate,        2,6-dichloro-4-(1,1,3,3 tetramethylbutyl)phenoxyacetate,        2,4-bis(1,1-dimethylpropyl)phenoxyacetate,        chlorodiphenylacetate, isobutyrate, monosuccinate,        (E)-2-methyl-2-butenoate (tigloate),        o-(methoxycarbonyl)benzoate, p-poly-benzoate, α-naphthoate,        nitrate, alkyl N,N,N′,N′-tetramethyl-phosphorodiamidate,        n-phenylcarbamate, borate, 2,4-dinitrophenylsulfenate); and    -   sulfonates (sulfate, methanesulfonate (mesylate),        benzylsulfonate, tosylate, triflate).        Compounds of the Invention

The present invention provides compounds of formulas I-IX, compositionsthat include such compounds, methods of their preparation, and methodsof their use. Specifically, the invention provides a compound of formulaI:

wherein

R¹ is (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy, aryl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkoxy, heteroaryl(C₁-C₆)alkoxy,aryl, heteroaryl, hydroxy, SR⁵, NR⁵R⁵, or absent;

R² is CH₂, carbonyl, SO₂, or OH;

L is CH₂, NR⁵, or O;

W¹-W⁶ are each independently C, N, O, S, or absent, and form a 5 or 6membered aryl, heterocycle, or heteroaryl ring;

W^(1′)-W^(6′) are each independently C, N, O, S, or absent, and form a 5or 6 membered aryl, heterocycle, or heteroaryl ring;

the dashed circles within the rings formed by W¹-W⁶ and W^(1′)-W^(6′)denote optional double bonds of the rings formed by W¹-W⁶ andW^(1′)-W^(6′);

R³ and R⁴ are each independently hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, aryl, heteroaryl, carboxy, cyano,nitro, halo, trifluoromethyl, trifluoromethoxy, SR⁵, SO₂N(R₅)₂, NR⁵R⁵,or COOR⁵;

each n is independently 0 to 4;

each R⁵ is independently H, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,(C₆-C₁₀)aroyl, aryl, aryl(C₁-C₆)alkyl, heteroaryl,heteroaryl(C₁-C₆)alkyl, or a nitrogen protecting group;

X is O, S, SO, SO₂, CH₂—O, CH₂—S, CH₂—NR⁵, NR, carbonyl, or a directbond;

D is S, SO, SO₂, P(O)OH, P(O)O(C₁-C₆)alkyl, P(O(C₁-C₆)alkyl)₂, C═N—OH,or carbonyl;

E is a direct bond, (C₁-C₆)allyl, (C₃-C₈)cycloalkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, or (C₃-C₈)heterocycle;

J is S, O, or NR⁵;

G, T, and Q are each independently H, (C₁-C₆)alkyl, or cyano;

any alkyl, amino, aryl, heteroaryl, or cycloalkyl is optionallysubstituted with 1 to about 5 (C₁-C₆)alkyl, (C₁-C₆)alkoxy, aryl,heteroaryl, aryl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl, nitro, halo,amino, or hydroxy groups;

or a pharmaceutically acceptable salt thereof;

provided that when L is CH₂ or 0, and R² is CH₂, R¹ is not (C₁-C₆)alkyl;

-   -   when L is O and R² is carbonyl, R¹ is not (C₁-C₆)alkyl; and    -   when L is NR, R² is CH₂.

The invention also provides a compound of formula II:

wherein

R¹ is H, (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkanoyloxy, aryl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl,aryl(C₁-C₆)alkoxy, heteroaryl(C₁-C₆)alkoxy, aryl, heteroaryl, hydroxy,nitro, cyano, halo, trifluoromethyl, trifluoromethoxy, SR⁵, NR⁵R⁵, orCO₂R⁵;

R² is CH₂, carbonyl, SO₂, or a direct bond;

L is CH₂, NR, O, S, SO, SO₂, or a direct bond;

W¹-W⁶ are each independently C, N, O, S, or absent, and form a 5 or 6membered aryl, heterocycle, or heteroaryl ring;

W^(1′)-W^(6′) are each independently C, N, O, S, or absent, and form a 5or 6 membered aryl, heterocycle, or heteroaryl ring;

the dashed circles within the rings formed by W¹-W⁶ and W^(1′)-W^(6′)denote optional double bonds of the rings formed by W¹-W⁶ andW^(1′)-W^(6′);

R³ and R⁴ are each independently hydroxy, (C₁-C₆)allyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, aryl, heteroaryl, carboxy, cyano,nitro, halo, trifluoromethyl, trifluoromethoxy, SR⁵, SO₂N(R₅)₂, NR⁵R⁵,or COOR⁵;

each n is independently 0 to 4;

each R⁵ is independently H, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,(C₆-C₁₀)aroyl, aryl, aryl(C₁-C₆)alkyl, heteroaryl,heteroaryl(C₁-C₆)alkyl, or a nitrogen protecting group;

X is CH₂—O, CH₂—NR⁵, CH₂—S, or carbonyl;

D is S, SO, SO₂, P(O)OH, P(O)O(C₁-C₆)alkyl, P(O(C₁-C₆)alkyl)₂, C═N—OH,or carbonyl;

E is a direct bond, (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, or (C₃-C₈)heterocycle;

J is S, O, or NR⁵;

G, T, and Q are each independently H, (C₁-C₆)alkyl, or cyano;

any alkyl, amino, aryl, heteroaryl, or cycloalkyl is optionallysubstituted with 1 to about 5 (C₁-C₆)alkyl, (C₁-C₆)alkenyl,(C₁-C₆)alkynyl, (C₁-C₆)alkoxy, aryl, heteroaryl, aryl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, nitro, halo, amino, or hydroxy groups;

or a pharmaceutically acceptable salt thereof.

The invention also provides a compound of formula III:

wherein

R¹ is H, (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkanoyloxy, aryl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl,aryl(C₁-C₆)alkoxy, heteroaryl(C₁-C₆)alkoxy, aryl, heteroaryl, hydroxy,nitro, cyano, halo, trifluoromethyl, trifluoromethoxy, SR⁵, NR⁵R⁵, orCO₂R⁵;

R² is CH₂, carbonyl, SO₂, or a direct bond;

L is CH₂, NR, O, S, SO, SO₂, or a direct bond;

W¹-W⁶ are each independently C, N, O, S, or absent, and form a 5 or 6membered aryl, heterocycle, or heteroaryl ring;

W^(1′)-W^(6′) are each independently C, N, O, S, or absent, and form a 5or 6 membered aryl, heterocycle, or heteroaryl ring;

the dashed circles within the rings formed by W¹-W⁶ and W^(1′)-W^(6′)denote optional double bonds of the rings formed by W¹-W⁶ andW^(1′)-W^(6′);

R³ and R⁴ are each independently hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, aryl, heteroaryl, carboxy, cyano,nitro, halo, trifluoromethyl, trifluoromethoxy, SR₅, SO₂N(R₅)₂, NR₅R₅,or COOR⁵;

each n is independently 0 to 4;

each R⁵ is independently H, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,(C₆-C₁₀)aroyl, aryl, aryl(C₁-C₆)alkyl, heteroaryl,heteroaryl(C₁-C₆)alkyl, or a nitrogen protecting group;

X is O, S, SO, SO₂, CH₂—O, CH₂—NR⁵, CH₂—S, N(R⁶), carbonyl, or a directbond;

D is P(O)OH, P(O)O(C₁-C₆)alkyl, P(O(C₁-C₆)alkyl)₂, C═N—OH, or carbonyl;

E is a direct bond, (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, or (C₃-C₈)heterocycle;

J is S, O, or NR⁵;

G, T, and Q are each independently H, (C₁-C₆)alkyl, or cyano;

any alkyl, amino, aryl, heteroaryl, or cycloalkyl is optionallysubstituted with 1 to about 5 (C₁-C₆)alkyl, (C₁-C₆)alkenyl,(C₁-C₆)alkynyl, (C₁-C₆)alkoxy, aryl, heteroaryl, aryl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)allyl, nitro, halo, amino, or hydroxy groups;

or a pharmaceutically acceptable salt thereof.

The invention also provides a compound of formula IV:

wherein

R¹ is H, (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkanoyloxy, aryl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl,aryl(C₁-C₆)alkoxy, heteroaryl(C₁-C₆)alkoxy, aryl, heteroaryl, hydroxy,nitro, cyano, halo, trifluoromethyl, trifluoromethoxy, SR⁵, NR⁵R⁵, orCO₂R⁵;

R² is CH₂, carbonyl, SO₂, or a direct bond;

L is CH₂, NR, O, S, SO, SO₂, or a direct bond;

W¹-W⁶ are each independently C, N, O, S, or absent, and form a 5 or 6membered aryl, heterocycle, or heteroaryl ring;

W^(1′)-W^(6′) are each independently C, N, O, S, or absent, and form a 5or 6 membered aryl, heterocycle, or heteroaryl ring;

the dashed circles within the rings formed by W¹-W⁶ and W^(1′)-W^(6′)denote optional double bonds of the rings formed by W¹-W⁶ andW^(1′)-W^(6′);

R³ and R⁴ are each independently hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, aryl, heteroaryl, carboxy, cyano,nitro, halo, trifluoromethyl, trifluoromethoxy, SR⁵, SO₂N(R₅)₂, NR⁵R⁵,or COOR⁵;

each n is independently 0 to 4;

each R⁵ is independently H, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,(C₆-C₁₀)aroyl, aryl, aryl(C₁-C₆)alkyl, heteroaryl,heteroaryl(C₁-C₆)alkyl, or a nitrogen protecting group;

X is NR⁶;

R⁶ is (C₁-C₆)alkanoyl, (C₆-C₁₀)aroyl, aryl, aryl(C₁-C₆)alkyl,heteroaryl, heteroaryl(C₁-C₆)alkyl, or a nitrogen protecting group;

D is S, SO, SO₂, P(O)OH, P(O)O(C₁-C₆)alkyl, P(O(C₁-C₆)alkyl)₂, C═N—OH,or carbonyl;

E is a direct bond, (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, or (C₃-C₈)heterocycle;

J is S, O, or NR⁵;

G, T, and Q are each independently H, (C₁-C₆)alkyl, or cyano;

any alkyl, amino, aryl, heteroaryl, or cycloalkyl is optionallysubstituted with 1 to about 5 (C₁-C₆)alkyl, (C₁-C₆)alkenyl,(C₁-C₆)alkynyl, (C₁-C₆)alkoxy, aryl, heteroaryl, aryl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, nitro, halo, amino, or hydroxy groups;

or a pharmaceutically acceptable salt thereof.

The invention also provides a compound of formula V:

wherein

R¹ is H, (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkanoyloxy, aryl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl,aryl(C₁-C₆)alkoxy, heteroaryl(C₁-C₆)alkoxy, aryl, heteroaryl,heterocycle, hydroxy, nitro, cyano, halo, trifluoromethyl,trifluoromethoxy, SR⁵, NR⁵R⁵, or CO₂R⁵;

R² is CH₂, carbonyl, SO₂, or a direct bond;

L is CH₂, NR⁵, O, S, SO, SO₂, or a direct bond;

W¹-W⁶ are each independently C, N, O, S, or absent, and form a 5 or 6membered aryl, heterocycle, or heteroaryl ring;

W^(1′)-W^(6′) are each independently C, N, O, S, or absent, and form a 5or 6 membered aryl, heterocycle, or heteroaryl ring;

the dashed circles within the rings formed by W¹-W⁶ and W^(1′)-W^(6′)denote optional double bonds of the rings formed by W¹-W⁶ and W^(1′)-W⁶;

R³ and R⁴ are each independently hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, aryl, heteroaryl, carboxy, cyano,nitro, halo, trifluoromethyl, trifluoromethoxy, SR⁵, SO₂N(R⁵)₂, NR⁵R⁵,or COOR⁵;

each n is independently 0 to 4;

m is 0 or 1;

each R⁵ is independently H, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,(C₆-C₁₀)aroyl, aryl, aryl(C₁-C₆)alkyl, heteroaryl,heteroaryl(C₁-C₆)alkyl, or a nitrogen protecting group;

X is O, S, SO, SO₂, CH₂—O, NR¹, carbonyl, or a direct bond;

Y is O or NR⁵;

D is S, SO, SO₂, P(O)OH, P(O)O(C₁-C₆)alkyl, P(O(C₁-C₆)alkyl)₂, C═N—OH,or carbonyl;

E is a direct bond, (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, or (C₃-C₈)heterocycle;

J is S, O, or NR⁵;

G, T, and Q are each independently H, (C₁-C₆)alkyl, or cyano;

any alkyl, amino, aryl, heteroaryl, or cycloalkyl is optionallysubstituted with 1 to about 5 (C₁-C₆)alkyl, (C₁-C₆)alkenyl,(C₁-C₆)alkynyl, (C₁-C₆)alkoxy, aryl, heteroaryl, aryl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, nitro, halo, amino, or hydroxy groups;

or a pharmaceutically acceptable salt thereof.

The invention also provides a compound of formula VI:

wherein

R¹ is H, (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkanoyloxy, aryl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl,aryl(C₁-C₆)alkoxy, heteroaryl(C₁-C₆)alkoxy, aryl, heteroaryl,heterocycle, hydroxy, nitro, cyano, halo, trifluoromethyl,trifluoromethoxy, SR⁵, NR⁵R⁵, or CO₂R⁵;

R¹ is CH₂, carbonyl, SO₂, or a direct bond;

L is CH₂, NR⁵, O, S, SO, SO₂, or a direct bond;

W¹-W⁶ are each independently C, N, O, S, or absent, and form a 5 or 6membered aryl, heterocycle, or heteroaryl ring;

W^(1′)-W^(6′) are each independently C, N, O, S, or absent, and form a 5or 6 membered aryl, heterocycle, or heteroaryl ring;

the dashed circles within the rings formed by W¹-W⁶ and W^(1′)-W^(6′)denote optional double bonds of the rings formed by W¹-W⁶ andW^(1′)-W^(6′);

R³ and R⁴ are each independently hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, aryl, heteroaryl, carboxy, cyano,nitro, halo, trifluoromethyl, trifluoromethoxy, SR⁵, SO₂N(R₅)₂, NR⁵R⁵,or COOR⁵;

each n is independently 0 to 4;

m is 0 or 1;

each R⁵ is independently H, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,(C₆-C₁₀)aroyl, aryl, aryl(C₁-C₆)alkyl, heteroaryl,heteroaryl(C₁-C₆)alkyl, or a nitrogen protecting group;

X is O, S, SO, SO₂, CH₂—O, NR⁵, carbonyl, or a direct bond;

Y is O or NR⁵;

D is S, SO, SO₂, P(O)OH, P(O)O(C₁-C₆)alkyl, P(O(C₁-C₆)alkyl)₂, C═N—OH,or carbonyl;

E is a direct bond, (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, or (C₃-C₈)heterocycle;

J is S, O, or NR⁵;

G, T, and Q are each independently H, (C₁-C₆)alkyl, or cyano;

any alkyl, amino, aryl, heteroaryl, or cycloalkyl is optionallysubstituted with 1 to about 5 (C₁-C₆)alkyl, (C₁-C₆)alkenyl,(C₁-C₆)alkynyl, (C₁-C₆)alkoxy, aryl, heteroaryl, aryl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, nitro, halo, amino, or hydroxy groups;

or a pharmaceutically acceptable salt thereof.

The invention also provides a compound of formula VII:

wherein

R¹ is H, (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkanoyloxy, aryl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl,aryl(C₁-C₆)alkoxy, heteroaryl(C₁-C₆)alkoxy, aryl, heteroaryl,heterocycle, hydroxy, nitro, cyano, halo, trifluoromethyl,trifluoromethoxy, SR⁵, NR⁵R⁵, or CO₂R⁵;

R² is CH₂, carbonyl, SO₂, or a direct bond;

L is CH₂, NR⁵, O, S, SO, SO₂, or a direct bond;

W¹-W⁶ are each independently C, N, O, S, or absent, and form a 5 or 6membered aryl, heterocycle, or heteroaryl ring;

W^(1′)-W^(6′) are each independently C, N, O, S, or absent, and form a 5or 6 membered aryl, heterocycle, or heteroaryl ring;

the dashed circles within the rings formed by W¹-W⁶ and W^(1′)-W^(6′)denote optional double bonds of the rings formed by W¹-W⁶ andW^(1′)-W^(6′);

R³ and R⁴ are each independently hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, aryl, heteroaryl, carboxy, cyano,nitro, halo, trifluoromethyl, trifluoromethoxy, SR⁵, SO₂N(R)₂, NR⁵R⁵, orCOOR⁵;

each n is independently 0 to 4;

m is 0 or 1;

each R⁵ is independently H, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,(C₆-C₁₀)aroyl, aryl, aryl(C₁-C₆)alkyl, heteroaryl,heteroaryl(C₁-C₆)allyl, or a nitrogen protecting group;

X is O, S, SO, SO₂, CH₂—O, NR⁵, carbonyl, or a direct bond;

D is S, SO, SO₂, P(O)OH, P(O)O(C₁-C₆)alkyl, P(O(C₁-C₆)alkyl)₂, C═N—OH,or carbonyl;

R⁷ is H, hydroxy, (C₁-C₆)allyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy, aryl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkoxy, heteroaryl(C₁-C₆)alkoxy,aryl, heteroaryl, heterocycle, halo, trifluoromethyl, trifluoromethoxy,NR⁵R⁵, or CO₂R⁵;

E is (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, or(C₃-C₉)heterocycle;

J is S, O, or NR⁵;

G, T, and Q are each independently H, (C₁-C₆)alkyl, or cyano;

any alkyl, amino, aryl, heteroaryl, or cycloalkyl is optionallysubstituted with 1 to about 5 (C₁-C₆)alkyl, (C₁-C₆)alkenyl,(C₁-C₆)alkynyl, (C₁-C₆)alkoxy, aryl, heteroaryl, aryl(C₁-C₆)alkyl,heteroalyl(C₁-C₆)alkyl, nitro, halo, amino, or hydroxy groups;

or a pharmaceutically acceptable salt thereof.

The invention also provides a compound of formula VIII:

wherein

R¹ is H, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, halo(C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, aryl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkoxy, heteroaryl(C₁-C₆)alkoxy,aryl, heteroaryl, heterocycle, hydroxy, nitro, cyano, halo,trifluoromethyl, trifluoromethoxy, SR⁵, NR⁵R⁵, or CO₂R⁵;

R² is CH₂, carbonyl, SO₂, or a direct bond;

L is CH₂, NR, O, S, SO, SO₂, or a direct bond;

W¹-W⁶ are each independently C, N, O, S, or absent, and form a 5 or 6membered aryl, heterocycle, or heteroaryl ring;

W^(1′)-W^(6′) are each independently C, N, O, S, or absent, and form a 5or 6 membered aryl, heterocycle, or heteroaryl ring;

the dashed circles within the rings formed by W¹-W⁶ and W^(1′)-W^(6′)denote optional double bonds of the rings formed by W¹-W⁶ andW^(1′)-W^(6′);

R³ and R⁴ are each independently hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, aryl, heteroaryl, carboxy, cyano,nitro, halo, trifluoromethyl, trifluoromethoxy, SR⁵, SO₂N(R₅)₂, NR⁵R⁵,or COOR⁵;

each n is independently 0 to 4;

m is 0 or 1;

each R⁵ is independently H, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,(C₆-C₁₀)aroyl, aryl, aryl(C₁-C₆)alkyl, heteroaryl,heteroaryl(C₁-C₆)alkyl, or a nitrogen protecting group;

X is O, S, SO, SO₂, CH₂—O, NR⁵, carbonyl, or a direct bond;

Y is C(R⁵) or N;

D is S, SO, SO₂, P(O)OH, P(O)O(C₁-C₆)alkyl, P(O(C₁-C₆)alkyl)₂, C═N—OH,or carbonyl;

R⁷ is H, hydroxy, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy, aryl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkoxy, heteroaryl(C₁-C₆)alkoxy,aryl, heteroaryl, heterocycle, halo, trifluoromethyl, trifluoromethoxy,NR⁵R⁵, or CO₂R⁵;

E is a direct bond, (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, or (C₃-C₈)heterocycle;

J is S, O, or NR⁵;

G, T, and Q are each independently H, (C₁-C₆)alkyl, or cyano;

any alkyl, amino, aryl, heteroaryl, or cycloalkyl is optionallysubstituted with 1 to about 5 (C₁-C₆)alkyl, (C₁-C₆)alkenyl,(C₁-C₆)alkynyl, (C₁-C₆)alkoxy, aryl, heteroaryl, aryl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, nitro, halo, amino, or hydroxy groups;

or a pharmaceutically acceptable salt thereof.

The invention also provides a compound of formula IX:

wherein

R¹ is H, (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkanoyloxy, aryl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl,aryl(C₁-C₆)alkoxy, heteroaryl(C₁-C₆)alkoxy, aryl, heteroaryl, hydroxy,nitro, cyano, halo, trifluoromethyl, trifluoromethoxy, SR⁵, NR⁵R⁵, orCO₂R⁵;

R² is CH₂, carbonyl, SO₂, or a direct bond;

L is CH₂, NR, O, S, SO, SO₂, or a direct bond;

W¹-W⁶ are each independently C, N, O, S, or absent, and form a 5 or 6membered aryl, heterocycle, or heteroaryl ring;

the dashed circles within the rings formed by W¹-W⁶ denote optionaldouble bonds of the ring formed by W¹-W⁶;

each n is independently 0 to 4 and the sum of n groups is not greaterthan 4;

each R³ and R⁴ are independently hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, aryl, heteroaryl, carboxy, cyano,nitro, halo, trifluoromethyl, trifluoromethoxy, SR⁵, SO₂N(R₅)₂, NR⁵R⁵,or COOR⁵;

or each n is 1 and R³ and R⁴ together form an ortho-fused aryl,heteroaryl, carbocycle, or heterocycle attached to two of W²-W⁶;

each R⁵ is independently H, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,(C₆-C₁₀)aroyl, aryl, aryl(C₁-C₆)alkyl, heteroaryl,heteroaryl(C₁-C₆)alkyl, or a nitrogen protecting group;

D is S, SO, SO₂, P(O)OH, P(O)O(C₁-C₆)alkyl, P(O(C₁-C₆)alkyl)₂, C═N—OH,carbonyl, or absent;

E is a direct bond, (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, or (C₃-C₈)heterocycle;

J is S, O, or NR⁵;

G, T, and Q are each independently H, (C₁-C₆)alkyl, or cyano;

any alkyl, amino, aryl, heteroaryl, heterocycle, carbocycle, orcycloalkyl is optionally substituted with 1 to about 5 (C₁-C₆)alkyl,(C₁-C₆)alkenyl, (C₁-C₆)alkynyl, (C₁-C₆)alkoxy, aryl, heteroaryl,aryl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl, nitro, halo, amino, or hydroxygroups;

or a pharmaceutically acceptable salt thereof.

The following definitions apply to compounds of each of formulas I-IX,unless otherwise noted.

One value of R¹ is (C₁-C₆)alkyl. A specific value of R¹ is methyl. R¹can also be ethyl, n-propyl, iso-propyl, or sec-butyl.

Another value for R¹ is (C₁-C₆)alkoxy. R¹ can also be methoxy orhydroxy.

Another value for R¹ is heteroaryl(C₁-C₆)alkoxy. Specific values of Rinclude pyridylmethyloxy, furanylmethyloxy, and thiophenylmethyloxy.

Another value for R¹ is halo(C₁-C₆)alkyl. A specific value of R¹ isbromopentyl.

Another value for R¹ is aryl(C₁-C₆)allyl. A specific value of R¹ isbenzyl.

Another value for R¹ is aryl. A specific value of R¹ is phenyl.

The group R¹ can be aryl substituted with 1 to about 5 (C₁-C₆)alkyl,(C₁-C₆)alkoxy, aryl, heteroaryl, aryl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, nitro, halo, amino, or hydroxy groups.Specifically, R¹ can be aryl substituted with methyl, methoxy,iso-propyl, nitro, halo, amino, or hydroxy.

Another value for R¹ is NR⁵R⁵. In one embodiment, each R⁵ canindependently be H or (C₁-C₆)alkyl. One specific value of R¹ isdiethylamino.

In one embodiment, R¹ is absent and R² is OH.

For the compounds of any one of formulas II-IX, a specific value for R¹is H. In formulas II-IX, R² and L can both be absent. In certainspecific embodiments of formulas II-IX, R¹ is H and R² and L are bothabsent.

One value for R² is CH₂. Another value for R² is carbonyl. A furthervalue for R² is SO₂.

One value for L is CH₂. Another value for L is NR⁵. Specific values forL include NR⁵ wherein R⁵ is H, (C₁-C₆)alkyl, or a nitrogen protectinggroup. Another specific value for L is O.

The rings formed by W¹-W⁶ can form a phenyl ring. The rings formed byW¹-W⁶ can also form a 6-membered heteroaryl ring, such a pyridyl ring.The rings formed by W¹-W⁶ can also form a 5-membered heteroaryl ring.Examples of such 5-membered heteroaryl rings include imidazole,thiazole, triazole, tetrazole, furan, and thiophene rings.

For the compounds of any of formulas I-VIII, the rings formed byW^(1′)-W^(6′) can form a phenyl ring. The rings formed by W^(1′)-W^(6′)can also form a 6-membered heteroaryl ring, such a pyridyl ring. Therings formed by W^(1′)-W^(6′) can also form a 5-membered heteroarylring. Examples of such 5-membered heteroaryl rings include imidazole,thiazole, triazole, tetrazole, furan, and thiophene rings.

The dashed circles within the ring formed by W¹-W⁶ can be two or threeconjugated double bonds. For the compounds of any of formulas I-VIII,the dashed circles within the ring formed by W^(1′)-W^(6′) can also betwo or three conjugated double bonds.

The value R³ can be absent (i.e., the value of n is 0). In certainembodiments, values for R³ include hydroxy, halo, amino, or combinationsthereof. R³ can also be hydroxyphenyl or halophenyl. In yet anotherembodiment, R³ can be SO₂N(R₅)₂. In one embodiment, each R⁵ of R³ is H.

The value R⁴ can be absent (i.e., the value of n is 0). In certainembodiments, values for R⁴ include hydroxy, halo, amino, or combinationsthereof. R⁴ can also be hydroxyphenyl or halophenyl. In yet anotherembodiment, R⁴ can be SO₂N(R₅)₂. In one embodiment, each R⁵ of R⁴ is H.

For the compounds of any of formulas I, III, and V-IX, X can be O. Otherspecific values for X include S, SO, SO₂, CH₂—O, CH₂—S, NR, CH₂—NR⁵,carbonyl, and a direct bond.

The attachment of the groups CH₂—O, CH₂—S, and CH₂—NR is ‘reversible’,i.e., the methylene group can be attached to either ring of formulas I,III, and V-IX. For example, the methylene group can be attached to thering formed by W¹-W⁶ or the ring formed by W^(1′)-W^(6′). Accordingly,the heteroatom of the recited group would then be attached to the otherring.

For the compounds of any of formulas I, III, and V-IX, when X is NR⁵, R⁵can be H. In other embodiments, R⁵ can be (C₁-C₆)alkyl, for example,methyl. In another embodiment, R⁵ can be aryl, for example, phenyl. Inyet another embodiment, R⁵ can be aryl(C₁-C₆)alkyl, for example, benzyl.In another embodiment, R⁵ can be a heteroaryl(C₁-C₆)alkyl, such aspyridylmethyl, imidazolylmethyl, thiazolylmethyl, triazolylmethyl,tetrazolylmethyl, furanylmethyl, or thiophenylmethyl. In yet anotherembodiment, R⁵ can be a nitrogen protecting group.

For the compounds of any of formulas I, III, and V-IX, when X isCH₂—NR⁵, R⁵ can be H. In other embodiments, R⁵ can be (C₁-C₆)alkyl, forexample, methyl. In another embodiment, R⁵ can be aryl, for example,phenyl. In yet another embodiment, R⁵ can be aryl(C₁-C₆)alkyl, forexample, benzyl. In another embodiment, R⁵ can be aheteroaryl(C₁-C₆)alkyl, such as pyridylmethyl, imidazolylmethyl,thiazolylmethyl, triazolylmethyl, tetrazolylmethyl, furanylmethyl, orthiophenylmethyl. In yet another embodiment, R⁵ can be a nitrogenprotecting group.

For the compounds of any of formulas I-II and IV-IX, one specific valueof D is S. Another specific value of D is SO. Yet another specific valueof D is SO₂.

For the compounds of any of formulas I-IX, D can be P(O)OH,P(O)O(C₁-C₆)alkyl, for example, P(O)OCH₃, or P(O(C₁-C₆)alkyl)₂, forexample, P(OCH₃)₂. In another embodiment, D can be C═N—OH. In yetanother embodiment, D can be carbonyl.

The variable E can be a direct bond. E can also be (C₁-C₆)alkyl, forexample, CH₂. In another embodiment, B can be (C₃-C₈)cycloalkyl, such ascyclohexyl, or a geminally-substituted cyclohexyl. E can also be(C₂-C₆)alkenyl, for example, 2-butenyl. Another value for E is(C₂-C₆)alkynyl. In a specific embodiment, E can be 2-butynyl. In otherembodiments, E can be (C₃-C₈)heterocycle, for example, piperidynyl. Thepiperidynyl can be an N-substituted piperidynyl linked to both itsneighboring groups at the 4-position.

The variable J can be S, O, or NR⁵. In one embodiment, J is NR⁵ and R⁵is H. In another embodiment, J can be NR⁵ and R⁵ can be (C₁-C₆)alkyl,for example, methyl. In another embodiment, J can be NR⁵ and R⁵ can be(C₁-C₆)alkanoyl, such as acetyl (—C(═O)CH₃). In yet another embodiment,J can be NR⁵ and R⁵ can be (C₆-C₁₀)aroyl, for example, benzoyl; aryl,for example, phenyl; aryl(C₁-C₆)alkyl, for example, benzyl; heteroaryl,for example, imidazolyl, thiazolyl, triazolyl, tetrazolyl, furanyl,thiophenyl, or pyridinyl; heteroaryl(C₁-C₆)alkyl, for example,imidazolylmethyl, thiazolylmethyl, triazolylmethyl, tetrazolylmethyl,furanylmethyl, thiophenylmethyl, or pyridinylmethyl; or a nitrogenprotecting group.

The variable G can be H. Another value for G is (C₁-C₆)alkyl, forexample, methyl. Yet another value for G is cyano.

The variable T can be H. Another value for T is (C₁-C₆)alkyl, forexample, methyl. Yet another value for T is cyano.

The variable Q can be H. Another value for Q is (C₁-C₆)alkyl, forexample, methyl. Yet another value for Q is cyano.

In one embodiment, G, T, and Q are each H.

For the compounds of formulas IV, X can be NR⁶, wherein R⁶ is(C₁-C₆)alkanoyl. In a specific embodiment, R⁶ is acetyl. R⁶ can also be(C₆-C₁₀)aroyl, for example, benzoyl; aryl, for example, phenyl;aryl(C₁-C₆)alkyl, for example, benzyl; heteroaryl, for example, pyridyl,imidazolyl, thiazolyl, triazolyl, tetrazolyl, furanyl, or thiophenyl;heteroaryl(C₁-C₆)alkyl, for example, pyridylmethyl, imidazolylmethyl,thiazolylmethyl, triazolylmethyl, tetrazolylmethyl, furanylmethyl, orthiophenylmethyl; or a nitrogen protecting group.

For the compounds of formulas V or VI, the value of m can be 0. In otherembodiments, the value of m can be 1.

For the compounds of formulas V or VI, Y can be O. In other embodiments,Y can be NR⁵. In one embodiment, Y is NR⁵, and R⁵ is H. In otherembodiments, Y can be NR⁵, and R⁵ can be (C₁-C₆)alkyl, for example,methyl; (C₁-C₆) alkanoyl, for example, acetyl; (C₆-C₁₀)aroyl, forexample, benzoyl; aryl, for example, phenyl; aryl(C₁-C₆)alkyl, forexample, benzyl; heteroaryl, for example, pyridyl, imidazolyl,thiazolyl, triazolyl, tetrazolyl, furanyl, or thiophenyl;heteroaryl(C₁-C₆)alkyl, for example, pyridylmethyl, imidazolylmethyl,thiazolylmethyl, triazolylmethyl, tetrazolylmethyl, furanylmethyl, orthiophenylmethyl; or a nitrogen protecting group.

For the compounds of formulas VII and VIII, R⁷ can be H. R⁷ can also behydroxy. Another value for R⁷ is (C₁-C₆)alkyl, for example, methyl.Another value for R⁷ is (C₂-C₆)alkenyl, for example, propenyl. Anothervalue for R⁷ is (C₂-C₆)alkynyl, for example, propynyl. Another value forR⁷ is halo(C₁-C₆)alkyl, for example, bromopentyl. Another value for R⁷is (C₁-C₆)alkoxy, for example, methoxy. Another value for R⁷ isaryl(C₁-C₆)allyl, for example, benzyl. Another value for R⁷ isheteroaryl(C₁-C₆)alkyl, for example, pyridylmethyl, imidazolylmethyl,thiazolylmethyl, triazolylmethyl, tetrazolylmethyl, furanylmethyl, orthiophenylmethyl. Another value for R⁷ is aryl(C₁-C₆)alkoxy, forexample, benzyloxy. Another value for R⁷ is heteroaryl(C₁-C₆)alkoxy,pyridylmethyloxy. Another value for R⁷ is aryl, for example, phenyl.Another value for R⁷ is heteroaryl, for example, pyridyl, imidazolyl,thiazolyl, triazolyl, tetrazolyl, furanyl, or thiophenyl. Another valuefor R⁷ is heterocycle, for example, piperidinyl.

For the compounds of formulas VII and VIII, R⁷ can also be halo, forexample, fluoro, chloro, bromo, or iodo. Yet another value for 17 istrifluoromethyl. Another value for R⁷ is trifluoromethoxy. Another valuefor R⁷ is NR⁵R⁵, wherein each R⁵ is H. Another value for R⁷ is NR⁵R⁵,wherein each R⁵ is (C₁-C₆)alkyl. Another value for R⁷ is NR⁵R⁵, whereineach R⁵ is methyl. Another value for R⁷ is NR⁵R⁵, wherein one R⁵ is Hand the other R⁵ of R⁷ is a nitrogen protecting group. Another value forR⁷ is CO₂R⁵. Another value for R⁷ is CO₂R⁵, wherein R⁵ is H. Anothervalue for R⁷ is CO₂R⁵, wherein R⁵ is (C₁-C₆)alkyl. Another value for R⁷is CO₂R⁵, wherein R⁵ is methyl. Another value for R⁷ is CO₂R⁵, whereinR⁵ is aryl. Yet another value for R⁷ is CO₂R⁵, wherein R⁵ is phenyl.

For the compounds of formula VIII, Y can be N or C(R⁵). In oneembodiment, Y is N. In another embodiment, Y is C(R⁵). In one specificembodiment, Y is C(R⁵), wherein R⁵ is H. In another embodiment, Y isC(R⁵), wherein R⁵ is (C₁-C₆)alkyl. In one specific embodiment, Y isC(R⁵), wherein R⁵ is methyl.

For the compounds of formula IX, each n can be 1 and R³ and R⁴ togethercan form an ortho-fused furanyl group. In another embodiment, each n is1 and R³ and R⁴ together form an ortho-fused1,2,3,4-tetrahydrobenzofuranyl group.

Methods of Use and Medical Indications

The invention also provides a pharmaceutical composition that includes acompound of any of formulas I-IX, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier. The pharmaceuticalcomposition can also include other therapeutic agents that arecompatible with the compound of the invention.

The invention also provides a compound of any of formulas I-IX, for usein medical therapy. The medical therapy can be for the treatment ofcancer, angiogenesis, cardiovascular disease, neurological disease,inflammation, eye disease, autoimmune disease, for regulatingcontraception, or other conditions that are affected by the regulationof MMPs.

The cancer can be pancreatic cancer, gastric cancer, lung cancer,colorectal cancer, prostate cancer, renal cell cancer, basal cellcancer, breast cancer, bone cancer, brain cancer, lymphoma, leukemia,melanoma, myeloma and other hematological cancers, and the like. Thecancer can be primary, metastatic, or both. The treatment of cancerusing a compound of the invention can affect angiogenesis.

The cardiovascular disease can be stroke, aneurysm, ischemia orreperfusion injury.

The neurological disease can be one that arises from at least one ofpainful neuropathy, neuropathic pain, diabetic neuropathy, drugdependence, drug withdrawal, depression, anxiety, movement disorders,tardive dyskinesia, cerebral infections that disrupt the blood-brainbarrier, meningitis, stroke, hypoglycemia, cardiac arrest, spinal cordtrauma, head trauma, and perinatal hypoxia. The neurological disease canalso be a neurodegenerative disorder. The neurological disease can beepilepsy, Alzheimer's disease, Huntington's disease, Parkinson'sdisease, multiple sclerosis, or amyotrophic lateral sclerosis, as wellas Alexander disease, Alper's disease, Ataxia telangiectasia, Battendisease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Canavandisease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakobdisease, Kennedy's disease, Krabbe disease, lewy body dementia,Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple SystemAtrophy, Pelizaeus-Merzbacher disease, Pick's disease, primary lateralsclerosis, Refsum's disease, Sandhoff disease, Schilder's disease,spinocerebellar ataxia (multiple types with varying characteristics),spinal muscular atrophy, Steele-Richardson-Olszewski disease, or tabesdorsalis.

The compounds of the invention can be used to treat conditions of theeye, including corneal wounds, glaucoma, dry eye disease, and maculadegeneration. The compounds can also be used to treat eye conditionsthat involve, are caused by, are effected by, or are exacerbated byMMP-9.

The compounds of the invention can be used to treat inflammation,wherein the inflammation involves connective tissue, airway tissue, orcentral nervous system tissue. The inflammation can be acute asthma,chronic asthma, allergic asthma, or chronic obstructive pulmonarydisease. In one embodiment, the inflammation is arthritis.

The compounds of the invention can be used to effect contraception,wherein the contraception occurs by inhibition of implantation.

The compounds of the invention can be used in medical therapy, whereinthe medical therapy is treatment of a skin disease.

The compounds of the invention can also be used in imaging, wherein theinhibitor can be modified to be detectable by imaging techniques; forpre- and post-operative treatments for removal of tumors; and incombination with any other chemotherapeutic modalities (biological andnon-biological).

The invention also provides the use of a compound of any of formulasI-IX, to prepare a medicament for treatment of cancer, angiogenesis,cardiovascular disease, neurological disease, inflammation, autoimmunedisease, or contraception. The medicaments can also be used to treat anyof the diseases or conditions discussed above.

The invention further provides a method to treat a disease comprisingcontacting a cell with a compound of formulas I-IX, wherein the compoundis effective to inhibit a matrix metalloproteinase. The invention alsoprovides a method to treat a subject in need thereof, comprisingadministering to the subject an effective amount of a matrixmetalloproteinase inhibitor of a compound of formulas I-IX. The matrixmetalloproteinase can be a gelatinase, collagenase, stromelysin,membrane-type My, or matrilysin. The matrix metalloproteinase can be,for example, MMP-2, MMP-9, or MMP-14. The matrix metalloproteinase canbe a human matrix metalloproteinase.

The matrix metalloproteinase inhibitor can be administered to thesubject in a pharmaceutically acceptable excipient. The subject can bean animal, for example, a mammal. The subject can be a human. Themethods employing the compound of formulas I-IX can be used to treat anyof the diseases or conditions discussed above.

A compound of formulas I-IX, or a pharmaceutically acceptable saltthereof, can be administered to a mammal (e.g., human) in conjunctionwith a chemotherapeutic agent, or a pharmaceutically acceptable saltthereof. Accordingly, a compound of formulas I-IX can be administered inconjunction with a chemotherapeutic agent to treat a disease, a tumor,or cancer.

According to one embodiment of the invention, the matrixmetalloproteinase can be contacted with the compound, e.g., a compoundof any one of formulas I-IX, in vitro. Alternatively, the matrixmetalloproteinase can be contacted with the compound, e.g., a compoundof any one of formulas I-IX, in vivo.

An important aspect of the invention is that a compound of formulas I-IXcan be selective for a particular matrix metalloproteinase over othermatrix metalloproteinases. This selectivity can provide a significantbenefit to treating the diseases and conditions discussed above becauseof the reduced dosage required for a given treatment.

Methods of Making the Compounds of the Invention.

Processes for preparing compounds of formulas I-IX and processes forpreparing intermediates useful for preparing compounds of formulas I-IXare provided as further embodiments of the invention. Intermediatesuseful for preparing compounds of formulas I-IX are also provided asfurther embodiments of the invention.

The compounds described herein can be prepared by any of the applicabletechniques of organic synthesis. Many such techniques are well known inthe art. However, many of the known techniques are elaborated inCompendium of Organic Synthetic Methods (John Wiley & Sons, New York)Vol. 1, Ian T. Harrison and Shuyen Harrison (1971); Vol. 2, Ian T.Harrison and Shuyen Harrison (1974); Vol. 3, Louis S. Hegedus and LeroyWade (1977); Vol. 4, Leroy G. Wade Jr., (1980); Vol. 5, Leroy G. WadeJr. (1984); and Vol. 6, Michael B. Smith; as well as March, J., AdvancedOrganic Chemistry, 3rd Edition, John Wiley & Sons, New York (1985);Comprehensive Organic Synthesis. Selectivity Strategy & Efficiency inModern Organic Chemistry, In 9 Volumes, Barry M. Trost, Editor-in-Chief,Pergamon Press, New York (1993); Advanced Organic Chemistry Part B:Reactions and Synthesis, 4th Ed.; Carey and Sundberg; KluwerAcademic/Plenum Publishers: New York (2001); Advanced Organic ChemistryReactions, Mechanisms, and Structure, 2nd Edition, March, McGraw Hill(1977); Protecting Groups in Organic Synthesis, 2nd Edition, Greene, T.W., and Wutz, P. G. M., John Wiley & Sons, New York (1991); andComprehensive Organic Transformations, 2nd Edition, Larock, R. C., JohnWiley & Sons, New York (1999).

As would be recognized by one skilled in the art, numerous modificationsand variations of the present invention are possible in light of theabove teachings. It is therefore to be understood that within the scopeof the appended claims, the invention may be practiced otherwise than asspecifically described herein.

Specific ranges, values, and embodiments provided herein are forillustrative purposes only and do not otherwise limit the scope of theinvention, as defined by the claims.

Pharmaceutical Formulations:

The compounds described herein can be administered as the parentcompound, a pro-drug of the parent compound, or an active metabolite ofthe parent compound.

The compounds of this invention can be formulated with conventionalcarriers and excipients, which can be selected in accord with ordinarypractice. Tablets will contain excipients, glidants, fillers, bindersand the like. Aqueous formulations are prepared in sterile form, andwhen intended for delivery by other than oral administration generallywill be isotonic. All formulations will optionally contain excipientssuch as those set forth in the Handbook of Pharmaceutical Excipients,5^(th) Ed.; Rowe, Sheskey, and Owen, Eds.; American PharmacistsAssociation; Pharmaceutical Press: Washington, D.C., 2006. Excipientsinclude ascorbic acid and other antioxidants, chelating agents such asEDTA, carbohydrates such as dextrin, hydroxyalkylcellulose,hydroxyalkylmethyl-cellulose, stearic acid and the like. The pH of theformulations ranges from about 3 to about 11, but is ordinarily about 7to 10.

While it is possible for the active ingredients to be administered aloneit may be preferable to present them as pharmaceutical formulations. Theformulations, both for veterinary and for human use, include at leastone active ingredient, as described herein, together with one or moreacceptable carriers therefor, and optionally other therapeuticingredients. The carrier(s) must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation andphysiologically innocuous to the recipient thereof.

The formulations include those suitable for the foregoing administrationroutes. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. Techniques and formulations generally are found in Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., (1985).Such methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also beadministered as a bolus, electuary or paste.

A tablet is made by compression or molding, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared by compressingin a suitable machine the active ingredient in a free-flowing form suchas a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered active ingredient moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and optionally are formulatedso as to provide slow or controlled release of the active ingredienttherefrom.

For administration to the eye or other external tissues e.g., mouth andskin, the formulations are preferably applied as a topical ointment orcream containing the active ingredient(s) in an amount of, for example,0.075 to 20% w/w (including active ingredient(s) in a range between 0.1%and 20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc.),preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. Whenformulated in an ointment, the active ingredients may be employed witheither a paraffinic or a water-miscible ointment base. Alternatively,the active ingredients may be formulated in a cream with an oil-in-watercream base.

If desired, the aqueous phase of the cream base may include, forexample, at least 30% w/w of a polyhydric alcohol, i.e. an alcoholhaving two or more hydroxyl groups such as propylene glycol, butane1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol(including PEG 400) and mixtures thereof. The topical formulations maydesirably include a compound which enhances absorption or penetration ofthe active ingredient through the skin or other affected areas. Examplesof such dermal penetration enhancers include dimethyl sulfoxide andrelated analogs.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner. While the phase may comprisemerely an emulsifier (otherwise known as an emulgent), it desirablycomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil. Preferably, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabilizer. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabilizer(s) make up theso-called emulsifying wax, and the wax together with the oil and fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the invention include Tween® 60, Span® 80, cetostearyl alcohol,benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodiumlauryl sulfate.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties. The cream should preferablybe a non-greasy, non-staining and washable product with suitableconsistency to avoid leakage from tubes or other containers. Straight orbranched chain, mono- or dibasic alkyl esters such as di-isoadipate,isocetyl stearate, propylene glycol diester of coconut fatty acids,isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters known asCrodamol CAP may be used, the last three being preferred esters. Thesemay be used alone or in combination depending on the propertiesrequired. Alternatively, high melting point lipids such as white softparaffin and/or liquid paraffin or other mineral oils are used.

Pharmaceutical formulations according to the present invention compriseone or more compounds of the invention together with one or morepharmaceutically acceptable carriers or excipients and optionally othertherapeutic agents. Pharmaceutical formulations containing the activeingredient may be in any form suitable for the intended method ofadministration. When used for oral use for example, tablets, troches,lozenges, aqueous or oil suspensions, dispersible powders or granules,emulsions, hard or soft capsules, syrups or elixirs may be prepared.Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsincluding sweetening agents, flavoring agents, coloring agents andpreserving agents, in order to provide a palatable preparation.

Tablets containing the active ingredient in admixture with non-toxicpharmaceutically acceptable excipient which are suitable for manufactureof tablets are acceptable. These excipients may be, for example, inertdiluents, such as calcium or sodium carbonate, lactose, lactosemonohydrate, croscarmellose sodium, povidone, calcium or sodiumphosphate; granulating and disintegrating agents, such as maize starch,or alginic acid; binding agents, such as cellulose, microcrystallinecellulose, starch, gelatin or acacia; and lubricating agents, such asmagnesium stearate, stearic acid or talc. Tablets may be uncoated or maybe coated by known techniques including microencapsulation to delaydisintegration and adsorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearatealone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsuleswhere the active ingredient is mixed with an inert solid diluent, forexample calcium phosphate or kaolin, or as soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium, such aspeanut oil, liquid paraffin or olive oil.

Aqueous suspensions of the invention contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropyl methylcelluose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol anhydride(e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension mayalso contain one or more preservatives such as ethyl or n-propylp-hydroxy-benzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oral suspensionsmay contain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents, such as those set forth above, and flavoringagents may be added to provide a palatable oral preparation. Thesecompositions may be preserved by the addition of an antioxidant such asascorbic acid.

Dispersible powders and granules of the invention suitable forpreparation of an aqueous suspension by the addition of water providethe active ingredient in admixture with a dispersing or wetting agent, asuspending agent, and one or more preservatives. Suitable dispersing orwetting agents and suspending agents are exemplified by those disclosedabove. Additional excipients, for example sweetening, flavoring andcoloring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, a mineral oil, such as liquid paraffin, ora mixture of these. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan monooleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate. Theemulsion may also contain sweetening and flavoring agents. Syrups andelixirs may be formulated with sweetening agents, such as glycerol,sorbitol or sucrose. Such formulations may also contain a demulcent, apreservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the invention may be in the form of asterile injectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butane-diol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 1 to 1000 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions (weight:weight). Thepharmaceutical composition can be prepared to provide easily measurableamounts for administration. For example, an aqueous solution intendedfor intravenous infusion may contain from about 3 to 500 μg of theactive ingredient per milliliter of solution in order that infusion of asuitable volume at a rate of about 30 mL/hour can occur.

Formulations suitable for administration to the eye include eye dropswherein the active ingredient is dissolved or suspended in a suitablecarrier, especially an aqueous solvent for the active ingredient. Theactive ingredient is preferably present in such formulations in aconcentration of 0.5 to 20%, advantageously 0.5 to 10% particularlyabout 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.1 to 500 microns (includingparticle sizes in a range between 0.1 and 500 microns in incrementsmicrons such as 0.5, 1, 30 microns, 35 microns, etc.), which isadministered by rapid inhalation through the nasal passage or byinhalation through the mouth so as to reach the alveolar sacs. Suitableformulations include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol or dry powder administration may beprepared according to conventional methods and may be delivered withother therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis of a given condition.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

The formulations are presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water for injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavoring agents.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefor.

Veterinary carriers are materials useful for the purpose ofadministering the composition and may be solid, liquid or gaseousmaterials which are otherwise inert or acceptable in the veterinary artand are compatible with the active ingredient. These veterinarycompositions may be administered orally, parenterally or by any otherdesired route.

Compounds of the invention can also be formulated to provide controlledrelease of the active ingredient to allow less frequent dosing or toimprove the pharmacokinetic or toxicity profile of the activeingredient. Accordingly, the invention also provided compositionscomprising one or more compounds of the invention formulated forsustained or controlled release.

Effective dose of active ingredient depends at least on the nature ofthe condition being treated, toxicity, whether the compound is beingused prophylactically (lower doses), the method of delivery, and thepharmaceutical formulation, and will be determined by the clinicianusing conventional dose escalation studies. It can be expected to befrom about 0.0001 to about 100 mg/kg body weight per day. Typically,from about 0.01 to about 10 mg/kg body weight per day. More typically,from about 0.01 to about 5 mg/kg body weight per day. More typically,from about 0.05 to about 0.5 mg/kg body weight per day. For example, thedaily candidate dose for an adult human of approximately 70 kg bodyweight will range from 1 mg to 1000 mg, preferably between 5 mg and 500mg, and may take the form of single or multiple doses.

One or more compounds of the invention (herein referred to as the activeingredients) are administered by any route appropriate to the conditionto be treated. Suitable routes include oral, rectal, nasal, topical(including buccal and sublingual), vaginal and parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, intrathecal andepidural), and the like. It will be appreciated that the preferred routemay vary with for example the condition of the recipient.

Mechanism of Action of Compounds of the Invention

A specific group of the compounds of the present invention, that can beactivated by zinc for nucleophilic substitution and that can form acovalent bond with a nucleophile of the matrix metalloproteinase,includes a thiirane ring. Another specific group of the compounds of thepresent invention, that can be activated by zinc for nucleophilicsubstitution and that can form a covalent bond with a nucleophile of thematrix metalloproteinase, includes an oxirane ring. In addition, aspecific nucleophile of the matrix metalloproteinase which can form acovalent bond with the group of the compounds of the present invention(e.g., thiirane or oxirane) is located at the amino acid residuecorresponding to residue 404 of the matrix metalloproteinase, whereinthe numbering is based on the active site general base for gelatinase A,which is observed in other MMPs. More specifically, the nucleophile is acarboxy (i.e., COO⁻) oxygen atom located at amino acid residuecorresponding to residue 404 of the matrix metalloproteinase, whereinthe numbering is based on the active site general base for gelatinase A,which is observed in other MMPs. See, FIG. 1.

Pharmaceutical Kits

Pharmaceutical kits useful in the present invention, which include atherapeutically effective amount of a pharmaceutical composition thatincludes a compound of component (a) and one or more compounds ofcomponent (b), in one or more sterile containers, are also within theambit of the present invention. Sterilization of the container may becarried out using conventional sterilization methodology well known tothose skilled in the art. Component (a) and component (b) may be in thesame sterile container or in separate sterile containers. The sterilecontainers or materials may include separate containers, or one or moremulti-part containers, as desired. Component (a) and component (b), maybe separate, or physically combined into a single dosage form or unit asdescribed above.

Such kits may further include, if desired, one or more of variousconventional pharmaceutical kit components, such as for example, one ormore pharmaceutically acceptable carriers, additional vials for mixingthe components, etc., as will be readily apparent to those skilled inthe art. Instructions, either as inserts or as labels, indicatingquantities of the components to be administered, guidelines foradministration, and/or guidelines for mixing the components, may also beincluded in the kit.

EXAMPLES

Several methods for the preparation of compounds of formulas I-IX areprovided in the Examples hereinbelow. These methods are intended toillustrate the nature of such compounds and preparations and are notintended to limit the scope of the compounds and applicable syntheticmethods of the invention.

Some abbreviations used herein include the following: Ac, acetyl; mCPBA,3-chloroperoxybenzoic acid; DMF, N,N-dimethylformamide; Me, methyl; Ms,methanesulfonyl; THF, tetrahydrofuran; MMP, matrix metalloproteinase;DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide(Me₂SO); PBS, phosphate buffer saline; FBS, fetal bovine serum.

Example 1 Design, Synthesis, and Evaluation of a Mechanism-BasedInhibitor for Gelatinase A

The first mechanism-based inhibitor for MMPs have been previouslydescribed (J. Am. Chem. Soc. 2000, 122, 6799-6800), which in chemistrymediated by the active site zinc ion selectively and covalently inhibitsMMP-2, MMP-3 and MMP-9. Computational analyses indicated that thisselectivity in inhibition of MMPs could be improved by design of newvariants of the inhibitor class. The syntheses of methyl2-(4-{4-[(2-thiiranylpropyl) sulfonyl]phenoxy}phenyl)-acetate (1.3) and2-(4-{4-[(2-thiiranylpropyl) sulfonyl]phenoxy}phenyl)acetic acid (1.4)are reported herein. The results of this Example show that compound 1.3serves as a mechanism-based inhibitor exclusively for MMP-2. Thismolecule should prove useful in delineating the functions of MMP-2 inbiological systems.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases withimportant pathological and physiological functions (Massova, I.; Kotra,L. P.; Fridman, R.; Mobashery, S. Matrix Metalloproteases: Structures,Evolution and Diversification, FASEB J 1998, 12, 1075; see also Can. J.Physiol. Pharmacol. 1999, 77, 465-480; J. Clin. Oncol. 2000, 18,1135-1149; Cell 2000, 100, 57-70; Nature Rev. 2002, 2, 563-572; NatureRev. 2002, 2, 727-739; Nature Rev. 2002, 3, 1-6; Nature Rev. 2003, 3,401-410; Nature Med. 2003, 9, 822-823; Nature Med. 2003, 9, 999-1000).

A total of 26 MMPs are known. Their unregulated and uncontrolledactivities have been associated with a number of disease processes,including neurological disorders, arthritis, cardiovascular diseases andcancer, neurological disorders, and others. Inhibition of MMPs as ameans to intervention of disease is highly sought (Nature Rev. 2002, 1,415-426; Current Opinion Chem. Biol. 1999, 3, 500-509; Chem. Rev. 1999,99, 2735-2776; Curr., Med. Chem. 2001, 8, 425-474; and Lee, M.; Fridman,R.; Mobashery, S. Chem. Soc. Rev. 2004, 33, 401-409). With very fewexceptions, the known inhibitors of MMPs are broad-spectrum molecules,designed to chelate the active-site zinc ions of these enzymes. Thisbroad breadth of activity has been problematic in clinical trials of MMPinhibitors, as the molecules tend to show serious side effects (Egebladand Werb, Nature Rev. Cancer 2002, 2, 161-174; Coussens et al. Science2002, 295, 2387-2392).

This Example is an investigation into selective inhibition ofgelatinases, MMP-2 and MMP-9 (also known as gelatinases A and B,respectively). The excessive and unregulated activities of these twoenzymes have been indicated in a number of cancer metastases (J. Surg.Res. 2003, 110, 383-392; Breast Cancer Res Treat 2003, 77, 145-155;Biochim. Biophys. Acta. 2004, 1705, 69-89; Gynecol. Oncol. 2004, 94,699-704; Eur. J. Surg. Oncol. 2004, 30, 560-4; and Mod. Pathol. 2004,17, 496-502).

Mobashery and co-workers disclosed for the first time a novel strategyin mechanism-based inhibition of MMP by a thiirane-containing inhibitor,where the thiirane sulfur first coordinates with the active-site zincion (J. Am. Chem. Soc. 2000, 122, 6799-6800). The coordinated thiiranepredisposes it to nucleophilic attack by the active site glutamate(Glu-404 in MMP-2) in these enzymes, a process that leads to covalentmodification of the enzyme and the attendant loss of activity. Thisprocess is depicted in FIG. 1 for a known member (compound 1.1) of thisclass of inhibitors (1.1→1.2).

Inhibitor 1.1 underwent the chemistry shown in FIG. 1 with three MMPs:MMP-2, MMP-3 and MMP-9. The goal of the experiments disclosed herein isto develop inhibitors of this class that show an even narrower spectrumof inhibition than that exhibited by compound 1.1. Specific interactionsat the bottom of the deep S1′ pocket of MMP-2 and MMP-9 were identifiedthat could be exploited with a structural variant of inhibitor 1.1 toenhance selectivity toward gelatinases (FIG. 2). Two such molecules, 1.3and 1.4, were designed by computational analyses. The syntheses of thesemolecules are reported herein. Furthermore, that compound 1.3 shows thepattern of slow-binding inhibition that leads to covalent chemistry onlywith MMP-2 is documented.

The syntheses of compounds 1.3 and 1.4 were accomplished according toScheme 1 below. An N,N-dimethyl glycine-promoted Ullmann couplingreaction between the commercially available aryl bromide 1.5 and phenol1.7, which was in turn prepared from 4-hydroxythiophenol (1.6) bychemoselective allylation, proceeded smoothly to give 1.8 in 65% yield.Oxidation of the sulfur and olefin moieties in 1.8 was achieved by theuse of an excess of m-CPBA (12 equiv.) to afford oxirane 1.9 in 92%yield, which was treated with thiourea to provide thiirane 1.3 in 77%yield.

Attempts at hydrolysis of the methyl ester of 1.3 to the correspondingcarboxylic acid 1.4 under various basic conditions were unsuccessful,probably due to the deprotonation of the acidic α-position to thesulfonyl moiety, followed by β-elimination of the thiolate. The methodof Mascaretti with the use of (Bu₃Sn)₂O in toluene at 80° C. was foundto be most effective for this conversion. Under these conditions, methylester 1.3 was converted to the corresponding tin ester, which wassubsequently hydrolyzed on C₁₈-reverse phase silica gel to afford thedesired carboxylic acid 1.4 in 65% yield (with an attendant 12% recoveryof 1.3).

The synthetic route to compounds 1.3 and 1.4 is different than thosereported for inhibitor 1.1. (J. Am. Chem. Soc. 2000, 122, 6799-6800; J.Org. Chem. 2004, 69, 3572-3573.) Syntheses of 1.1 began from thecommercially available diphenyl ethers 1.11 or 1.12 illustrated below.The synthetic route of Scheme 1 allows more flexibility in creatingstructural diversity in the two ring systems and should find moregeneral applicability for entries into this molecular class of versatileenzyme inhibitors.

Compounds 1.3 and 1.4 were evaluated with a representative set of MMPs.As shown in Table 1, compound 1.4 inhibits MMP-2 with a K; of 460 iiM.Whereas compound 1.3 exhibits dissociation constants for MMP-2 and -9 inthe low nanomolar levels, it behaves as a slow-binding inhibitor thatleads to mechanism-based inhibition only with MMP-2. Therefore, compound1.3 is a selective and potent mechanism-based inhibitor of MMP-2(gelatinase A). Both compounds are merely poor competitive inhibitors(micromolar) of the other MMP that were tested. Hence, high selectivityin inhibition of MMP-2 has been achieved.

TABLE 1 Kinetic Parameters for Competitive Inhibition of MMPs by theSynthetic Inhibitors K_(i) (nM) 1.3 1.4 MMP-2  50 ± 14^(a) 460 ± 30MMP-9  40 ± 2 (4.1 ± 0.2) × 10³ MMP-14_(cat) 590 ± 70 (5.3 ± 0.3) × 10⁴MMP-1 (1.1 ± 0.2) × 10⁴ (4.5 ± 0.9) × 10³ MMP-3 (8.7 ± 0.5) × 10³ (5.4 ±1.0) × 10⁵ MMP-7 1.3 × 10⁴ (2.5 ± 0.1) × 10⁵ ^(a)Parameters forslow-binding component of inhibition: k_(on) = (1.2 ± 0.3) □ 10⁴ M⁻¹s⁻¹,k_(off) = (6.2 ± 0.7) × 10⁻⁴ s⁻¹.

Superimposition of the X-ray structures for MMP-2 and MMP-9 reveals someimportant differences (J. Biol. Chem. 2003, 278, 51646-51653). Midwaythrough the S1′ loop, an arginine residue is present in MMP-9, asopposed to a threonine in MMP-2. The pocket of MMP-9 appears to be moreconstricted than that of MMP-2, as the backbone of the S1′ loop of MMP-9is about 2-3 Å inward. This could potentially lead to unfavorable stericinteraction for the methyl moiety of compound 1.3 in MMP-9.

Methods: Enzyme kinetics were performed as previously described (J. Am.Chem. Soc. 2000, 122, 6799-6800).

Experimental Procedures and Data for Compounds of Example 1

4-(Allylthio)phenol (1.7). To a stirred solution of 4-hydroxythiophenol(1.6) (4.30 g, 34.1 mmol) in DMF (25 mL) were added K₂CO₃ (4.71 g, 34.1mmol) and allyl bromide (3.09 mL, 34.1 mmol) at ice-water temperature,and the mixture was stirred for 15 minutes, prior to stirring overnightat room temperature. After the addition of 1 M aqueous HCl, the mixturewas extracted with ether (3×). The combined organic layer was washedwith water and brine, dried over MgSO₄, and concentrated under reducedpressure. The resultant residue was purified by silica gel columnchromatography (ethyl acetate/hexane=1/10 to 1/6) to give 1.7 (5.74 g,70%) as a white semi-solid. The ¹H and ¹³C NMR spectra and mass spectrumwere identical to the reported values (Tetrahedron 1994, 50,10321-10330).

Methyl 2-{4-[4-(Allylthio)phenoxy]phenyl}acetate (1.8). A mixture of 1.5(1.51 g, 6.59 mmol), 7 (1.64 g, 9.88.mmol), Cs₂CO₃ (4.30 g, 13.2 mmol),N, N-dimethylglycine hydrochloride salt (276 mg, 1.98 mmol), CuI (125mg, 0.659 mmol), and degassed 1,4-dioxane (14 mL) was heated at 90° C.for 22 hours under a nitrogen atmosphere. After dilution with water, themixture was extracted with ethyl acetate. The combined organic layer waswashed with water and brine, dried over Na₂SO₄, and concentrated underreduced pressure. The resultant residue was purified by silica gelcolumn chromatography (ethyl acetate/hexane=1/12) to give 1.8 (1.35 g,65%) as a pale yellow semi-solid. ¹H NMR (300 MHz, CDCl₃): δ 3.49 (d,2H, J=7.2 Hz), 3.61 (s, 2H), 3.71 (s, 3H), 5.03-5.10 (m, 2H), 5.86 (m,1H), 6.91-6.97 (m, 4H), 7.23-7.26 (m, 2H), 7.32-7.35 (m, 2H); ¹³C NMR(125 MHz, CDCl₃): δ 38.5, 40.3, 52.1, 117.5, 119.0, 119.1, 129.0, 129.3,130.6, 132.9, 133.7, 156.0, 156.3, 172.0; HRMS (FAB) calcd for C₁₈H₁₈O₃S(M⁺) 314.0977, found 314.0986.

Methyl 2-(4-{4-[(2-Oxiranylpropyl)sulfonyl]phenoxy}phenyl)-acetate(1.9). To a stirred solution of 1.8 (500 mg, 1.59 mmol) in CH₂Cl₂ (20mL) was added m-CPBA (ca. 70%, 4.7 g, 19.1 mmol) at ice-watertemperature, and the mixture was subsequently stirred at roomtemperature for eight days. With ice-cooling, the reaction was quenchedwith a saturated Na₂S₂O₃ solution, followed by saturated NaHCO₃solution, and the mixture was extracted with ethyl acetate (3×). Thecombined organic layer was washed with saturated Na₂S₂O₃ solution,saturated NaHCO₃ solution, water and brine, dried over Na₂SO₄, andconcentrated under reduced pressure. The resultant residue was purifiedby silica gel column chromatography (ethyl acetate/hexane=1/2 to 2/3) togive 1.9 (528 mg, 92%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃): δ2.47 (dd, 1H, J=5.0, 2.0 Hz), 2.82 (m, 1H), 3.26-3.33 (m, 3H), 3.65 (s,2H), 3.72 (s, 3H), 7.03-7.05 (m, 2H), 7.08-7.10 (m, 2H), 7.32-7.34 (m,2H), 7.86-7.88 (m, 2H); ¹³C NMR (125 MHz, CDCl₃): δ 40.3, 45.8, 52.1,59.6, 117.6, 120.6, 130.5, 130.9, 131.1, 132.4, 153.8, 162.8, 171.8;HRMS (FAB) calcd for C₈H₁₈O₆S (M⁺) 362.0824, found 362.0829.

Methyl 2-(4-{4-[(2-Thiiranylpropyl)sulfonyl]phenoxy}phenyl)-acetate(1.3). To a stirred solution of 1.9 (500 mg, 1.38 mmol) in MeOH—CH₂Cl₂(10:1, 11 mL) was added thiourea (262 mg, 3.45 mmol) at roomtemperature, and the mixture was stirred overnight. After concentrationunder reduced pressure, the residue was dissolved in ethyl ether. Theethyl ether solution was washed with water and brine, dried over MgSO₄,and concentrated under reduced pressure. The residue was purified bysilica gel column chromatography (ethyl acetate/hexane=2/5) to give 1.3(400 mg, 77%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃): δ 2.15 (dd,1H, J=5.5, 2.0 Hz), 2.53 (dd, 1H, J=6.5, 2.0 Hz), 3.05 (m, 1H), 3.17(dd, 1H, J=14.5, 7.0 Hz), 3.51 (dd, 1H, J=14.5, 5.5 Hz), 3.65 (s, 2H),3.72 (s, 3H), 7.03-7.05 (m, 2H), 7.08-7.10 (m, 2H), 7.33-7.34 (m, 2H),7.85-7.86 (m, 2H); ¹³C NMR (125 MHz, CDCl₃): δ 24.2, 26.0, 40.3, 52.1,62.6, 117.7, 120.5, 130.7, 130.9, 131.1, 131.9, 153.8, 162.8, 171.8;HRMS (FAB) calcd for C₁₈H₁₉O₅S₂ (M+H⁺) 379.0674, found 379.0645.

2-(4-{4-[(2-Thiiranylpropyl)sulfonyl]phenoxy}phenyl)acetic acid (1.4).To a stirred solution of 3 (312 mg, 0.83 mmol) in toluene (11 mL) wasadded bis(tributyltin)oxide (1.05 mL, 2.06 mmol) at room temperature,and the mixture was stirred at 80° C. for 12 hours. The solution wascooled to room temperature and was concentrated to dryness under reducedpressure. The residue was dissolved in acetonitrile, and the solutionwas washed with hexane (3×) and concentrated under reduced pressure toleave the crude tin ester 1.10 (532 mg) as a pale-yellow oil.Subsequently, 1.10 was passed through a C₁₈-reverse phase silica gel pad(ODS silica gel 20 g, washed with water, 1:2 water/acetonitrile andacetonitrile) to afford a mixture of 1.3 and 1.4, which was purified bysilica gel column chromatography (chloroform/methanol=30/1 to 10/1) togive 1.4 (195 mg, 65%) as a white solid with the recovery of some of 1.3(38 mg, 12%). Compound 1.10: ¹H NMR (300 MHz, CDCl₃): δ 0.90 (t, 9H,J=7.2 Hz), 1.23-1.38 (m, 12H), 1.54-1.64 (m, 6H), 2.16 (dd, 1H, J=5.4,1.8 Hz), 2.54 (dd, 1H, J=6.0, 1.8 Hz), 3.07 (m, 1H), 3.15 (dd, 1H,J=13.8, 7.8 Hz), 3.54 (dd, 1H, J=13.8, 5.1 Hz), 3.64 (s, 2H), 7.03 (m,2H), 7.08 (m, 2H), 7.35 (m, 2H), 7.86 (m, 2H); Mass (FAB): m/z 655(M+H⁺); Rf value=0.3 (chloroform/methanol=10/1). Compound 1.4: mp133-134° C.; ¹H NMR (500 MHz, CDCl₃): δ 2.16 (d, 1H, J=4.0 Hz), 2.54 (d,1H, J=5.5 Hz), 3.06 (m, 1H), 3.19 (dd, 1H, J=14.0, 8.0 Hz), 3.52 (dd,1H, J=14.0, 6.0 Hz), 3.68 (s, 2H), 7.05 (br d, 2H, J=8.5 Hz), 7.10 (brd, 2H, J=8.5 Hz), 7.35 (br d, 2H, J=8.5 Hz), 7.86 (br d, 2H, J=8.5 Hz);¹³C NMR (125 MHz, CDCl₃): δ 24.2, 26.0, 40.2, 62.6, 117.8, 120.5, 130.2,130.7, 131.3, 132.0, 154.1, 162.7, 177.1; HRMS (FAB) calcd forC₁₇H₁₇O₅S₂ (M+H⁺) 365.0517, found 365.0495; Rf value=0.2(chloroform/methanol=10/1).

Computational Procedures. The X-ray structure of MMP-2 provided theCartesian coordinates for the molecular docking study (RCSB code 1 QIB).The Sybyl program (Tripos Inc., St. Louis, Mo.) was used for themanipulation and visualization of all structures and for the protonationof the bound ligand. AM1-BCC charges were computed for the ligand usingthe antechamber module from the AMBER 7 suite of programs (Case et al.,AMBER 7 ed.; University of California: San Francisco, 2002). The ligandwas docked into the active site of MMP-2 using a Lamarekian geneticalgorithm as implemented in the AutoDock 3.04 program (Morris et al. J.Comp. Chem. 1998, 19, 1639-1662). Parameters for the docking runs weresimilar to those used previously (Morris et al. J. Comp. Chem. 1998, 19,1639-1662), except for the following differences: the quaternion step,the translation step, and the torsion step were set to 0.2, 5, and 5,respectively. The number of evaluations was increased to 2.5×10⁷ from250,000 and the ligand was fully flexible during the docking runs.

Example 2 Potent Mechanism-Based Inhibitors For MatrixMetalloproteinases

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases thatplay important roles in physiological and pathological conditions. Bothgelatinases (MMP-2 and MMP-9) and membrane-type 1 MMP (MMP-14) areimportant targets for inhibition since their roles in various diseases,including cancer, have been well established. This Example describes aset of mechanism-based inhibitors that show high selectivity togelatinases and MMP-14 (inhibitor 2.3) and to only MMP-2 (inhibitors 2.5and 2.7). These molecules bind to the active sites of these enzymes,initiating a slow-binding profile for the onset of inhibition, whichleads to covalent enzyme modification. The full kinetic analysis for theinhibitors is reported. These are nanomolar inhibitors (K_(i)) for theformation of the non-covalent enzyme-inhibitor complexes. The onset ofslow-binding inhibition is rapid (k_(on) of 10² to 10⁴ M⁻¹s⁻¹) and thereversal of the process is slow (k_(off) of 10⁻³ to 10⁻⁴ s⁻¹). However,with the onset of covalent chemistry with the best of these inhibitors(e.g., inhibitor 2.3), very little recovery of activity (<10%) was seenover 48 hours of dialysis. We previously reported that broad-spectrumMMP inhibitors like GM6001 (hydroxamate inhibitor) enhanceMT1-MMP-dependent activation of pro-MMP-2 in the presence of TIMP-2.Herein, we show that inhibitor 2.3, in contrast to GM6001, had no effecton pro-MMP-2 activation by MT1-MMP. Furthermore, inhibitor 2.3 reducedtumor cell migration and invasion in vitro. These results show thatthese new inhibitors are promising candidates for selective inhibitionof MMPs in animal models of relevant human diseases.

Extracellular proteolysis is an essential aspect of both physiologicaland pathological processes. Several enzyme families have been implicatedin extracellular proteolysis, of which the matrix metalloproteinases(MMPs) constitute an important group. The MMPs are zinc-dependentendopeptidases that play key roles in embryonic development,neurological processes, wound healing, angiogenesis, arthritis,cardiovascular diseases and cancer, just to mention a few. In cancer,for instance, MMPs are implicated at all stages of tumor progression,including tumor growth, angiogenesis, and metastasis (Egeblad and Werb,(2002) Nat Rev Cancer 2, 161-174). Two MMPs, gelatinases A and B (MMP-2and MMP-9, respectively), are highly expressed in human cancer and adirect relationship between cancer progression and gelatinase expressionand activity has been well established in many studies (McCawley andMatrisian, (2000) Mol Med Today 6, 149-156). As tumors manifest highlevels of gelatinase activity, inhibitors specific for the gelatinasesare highly sought.

In the past eight years there have been numerous approaches aimed attargeting MMP activities in tumors and several clinical trials werecarried out to test the efficacy of various inhibitors. Unfortunately,the results of these trials were disappointing due to the lack of anobjective clinical response and undesired side effects. Many reasonshave been postulated for these effects but at the core of the problemremains the issue of inhibitor selectivity (Pavlaki, M. and Zucker, S.(2003) Cancer Metastasis Rev 22, 177-203; Coussens, L. M., Fingleton,B., and Matrisian, L. M. (2002) Science 295, 2387-2392). Indeed,virtually all MMP inhibitors tested so far have been broad-spectruminhibitors, designed around chelation of the active site zinc ion(Skiles et al. (2004) Curr Med Chem 11, 2911-2977) and their spectrum ofinhibition includes, in addition to MMPs, other metalloenzymes. Becausetargeting gelatinases remains to be of great promise in cancer therapy(Matrisian et al. (2003) Cancer Res 63, 6105-6109), efforts aimed atdeveloping better and selective gelatinase inhibitors continue.

A mere handful of selective inhibitors for MMPs have been reported inthe literature (for a review see: Brown, S., Meroueh, S. O., Fridman,R., and Mobashery, S. (2004) Curr Top Med Chem 4, 1227-1238). The designand properties of inhibitor 2.1 (Scheme 2) has been previouslydescribed. Inhibitor 2.1 is a selective mechanism-based inhibitor forgelatinases. This compound binds to the active sites of MMP-2 and MMP-9with the thiirane moiety coordinating with the zinc ion. Thiscoordination to the active site metal ion activates the thiirane ringfor opening by the nucleophilic attack of the active site glutamate inthese enzymes (FIG. 3 a). A unique property of this inhibitor is that onbinding to the active site zinc ion a pattern of slow-binding forinhibition sets in, leading to a rapid process for the on-set ofinhibition with an attendant slow process for recovery from slow-bindingat the non-covalent stage of inhibition. This non-covalent inhibitedspecies leads to covalent inhibition by modification of the glutamate.

The synthetic procedures for compounds 2.1-2.7 are given in theExperimental Procedures section below.

Inhibitor 2.1, the prototype of this type of novel mechanism-basedinhibitor for gelatinases, is showing promise in mouse models fordiseases involving gelatinases (Gu, Z., Cui, J., Brown, S., Fridman, R.,Mobashery, S., Strongin, A. Y., and Lipton, S. A. (2005) J. Neurosci.25, 6401-6408; Kruger, A., Arlt, M. J., Gerg, M., Kopitz, C., Bemardo,M. M., Chang, M., Mobashery, S., and Fridman, R. (2005) Cancer Res. 65,3523-3526). The poor solubility of this inhibitor in aqueous medium,however, is a limitation of the molecule. The compounds designed anddescribed in this Example provide increased aqueous solubility overinhibitor 2.1. Furthermore, the concept behind the inhibitor design intargeting other MMPs has been explored in this Example.

A computational model of the inhibitor bound in the active site of MMP-2within the constraints of the data from X-ray absorption spectroscopyhas been generated; see FIG. 2B; (Klcifeld, O., Kotra, L. P., Gervasi,D. C., Brown, S., Bernardo, M. M., Fridman, R., Mobashery, S., and Sagi,I. (2001) J. Biol. Chem. 276, 17125-17131). This model for inhibition ofinhibitor 2.1 led the way in exploration of the next generation of thistype of M inhibitor. The possibility for specific electrostaticinteractions near the terminal phenyl group in inhibitor 2.1 bound tothe active site of MMP-2 was anticipated for judiciously designedchemical functionalities into the molecular template of compound 2.1.

Three new functional groups are introduced in MMP inhibitors in thisExample, the methylsulfonamide (compounds 2.2 and 2.3), the nitro(compounds 2.4 and 2.5) and the acetamide (compounds 2.6 and 2.7), atthe terminal phenyl ring system to exploit these electrostaticinteractions (Scheme 52). It was expected that these molecules wouldimprove the solubility in aqueous solutions, while exhibiting highselectivity in inhibition toward gelatinases and the membrane-anchoredMMP, MT1-MMP (MMP-14), which all share a deep S1′ binding site. As willbe described herein, these expectations have been borne out, makingthese inhibitors valuable tools in studies of the functions of MMPs indisease processes. Furthermore, oxirane variants of these molecules(compounds 2.2, 2.4, and 2.6) have been prepared. The fact that theoxirane variants are either poor inhibitors or demonstrate no observableinhibitory properties toward MMPs underscore the importance of thethiirane group for this inhibitor class.

Experimental Procedures:

Synthesis—¹H and ¹³C NMR spectra were recorded on either a VarianUnityPlus 300 MHz or a Varian INOVA 500 MHz spectrometer. Chemicalshifts are reported in ppm from tetramethylsilane on the

 scale. Mass spectra were recorded on a JEOL JMS-AX505HA and aFinnigan-MAT 8430 high-resolution magnetic sector mass spectrometers.For silica gel column chromatography, EMD Silica gel 60 was employed.Thin-layer chromatography was performed with Whatman 0.25 mm silica gel60-F plates. All other reagents were purchased from Aldrich ChemicalCompany, Lancaster or Across Organics.

4-(Allylthio)phenol. To a stirred solution of 4-hydroxythiophenol (4.30g, 34.1 mmol) in DMF (25 ml) were added K₂CO₃ (4.71 g, 34.1 mmol) andallyl bromide (3.09 ml, 34.1 mmol) at ice-water temperature, and themixture was stirred for 15 minutes, prior to stirring overnight at roomtemperature. After the addition of 1 M aqueous HCl, the mixture wasextracted with ether (3×). The combined organic layer was washed withwater and brine, dried over MgSO₄, and concentrated under reducedpressure. The resultant residue was purified by silica gel columnchromatography (ethyl acetate/hexane, 1/10 to 1/6) to give 2.9 (5.74 g,70%) as a white semi-solid. The ¹H and ¹³C NMR spectra and mass spectrumwere identical to the reported values (Goux, C., Lhoste, P., and Sinou,D. (1994) Tetrahedron 50, 10321-10330).

1-Allylthio-4-(4-nitrophenoxy)benzene. To a stirred solution of 2.9(3.46 g, 20.8 mmol) in DMF (100 ml) were added cesium carbonate (10.2 g,31.2 mmol) and 1-fluoro-4-nitrobenzene (2.10) (2.94 g, 20.8 mmol) atroom temperature, and the mixture was stirred at the same temperaturefor 2 days. After dilution with water, the mixture was extracted intohexane (3×). The combined organic layer was washed with water and brine,dried over Na₂SO₄, and concentrated under reduced pressure to give 2.11(5.32 g, 89%) as a pale yellow oil. ¹H NMR (300 MHz, CDCl₃): δ 3.55 (dt,2H, J=6.9, 1.2 Hz), 5.10 (dt, 1H, J=10.2, 1.2 Hz), 5.13 (dt, 1H, J=17.1,1.2 Hz), 5.88 (ddt, 1H, J=17.1, 10.2, 6.9 Hz), 6.98-7.04 (m, 4H),7.38-7.43 (m, 2H), 8.18-8.22 (m, 2H); ¹³C NMR (125 MHz, CDCl₃): δ 37.8,117.1, 117.9, 120.9, 126.0, 132.3, 132.5, 133.4, 142.7, 153.4, 163.1;HRMS (FAB) calcd for C₁₅H₁₃NO₃S (M⁺) 287.0616, found 287.0593.

1-Allylthio-4-[4-(methanesulfonamido)phenoxy]benzene. To a stirredsolution of 2.11 (636 mg, 2.21 mmol) in THF (22 ml) were added aceticacid (2.54 ml, 44.2 mmol) and zinc powder (5.80 g, 88.4 mmol) at roomtemperature, and the suspension was stirred for 30 min (an exothermicreaction). After dilution with ethyl acetate, the mixture was filteredthrough Celite. The filtrate was washed with saturated NaHCO₃ and brine,dried over Na₂SO₄, and concentrated under reduced pressure to give acrude 2.12 (577 mg) as an orange oil, which was employed in the nextreaction without purification.

To a stirred solution of 2.12 (577 mg) in CH₂Cl₂ (10 ml) were addedpyridine (894 μL, 11.1 mmol) and methanesulfonyl chloride (205 μL, 2.65mmol) at ice-water temperature. After 15 min, the mixture was warmed toroom temperature and the stirring was continued for an additional 2 h.Subsequent to the addition of saturated NaHCO₃, the mixture wasextracted with ethyl acetate (3x). The combined organic layer was washedwith 1 M aqueous HCl, saturated NaHCO₃ solution and brine, dried overNa₂SO₄, and concentrated under reduced pressure. The resultant residuewas purified by silica gel column chromatography (CH₂Cl₂) to give 2.13(662 mg, 89% from 2.11) as a pale red solid. Compound 2.12: ¹H NMR (300MHz, CDCl₃): δ 3.45 (br.d, 2H, J=7.2 Hz), 3.59 (br.s, 2H), 5.01-5.06 (m,2H), 5.84 (ddt, 1H, J=17.1, 9.6, 6.9 Hz), 6.66-6.70 (m, 2H), 6.83-6.88(m, 4H), 7.29-7.32 (m, 2H). Compound 2.13: ¹H NMR (300 MHz, CDCl₃): δ3.01 (s, 3H), 3.50 (dt, 2H, J=7.2, 1.2 Hz), 5.04-5.11 (m, 2H), 5.86(ddt, 1H, J=16.8, 10.2, 6.9 Hz), 6.67 (br.s, 1H), 6.90-6.95 (m, 2H),6.96-7.01 (m, 2H), 7.20-7.26 (m, 2H), 7.32-7.37 (m, 2H); ¹³C NMR (75MHz, CDCl₃): □ 38.4, 39.3, 117.5, 119.3, 119.9, 123.8, 130.2, 132.0,132.9, 133.8, 155.2, 156.1; HRMS (FAB) calcd for C₁₆H₁₇NO₃S₂ (NO335.0650, found 335.0639.

4-(4-Acetamidophenory)-1-allylthiobenzene. To a stirred solution of 2.12(794 mg), which was prepared from 2.11 (830 mg, 2.89 mmol) in the samemanner as described for compound 2.13, in CH₂Cl₂ (15 ml) were addedpyridine (500 mL, 6.18 mmol) and acetic anhydride (292 μL, 3.09 mmol) atice-water temperature, and the mixture was stirred at the sametemperature for 1 h. Subsequent to the addition of saturated NaHCO₃, themixture was extracted with ethyl acetate (3×). The combined organiclayer was washed with 1 M aqueous HCl, saturated NaHCO₃ solution andbrine, dried over Na₂SO₄, and concentrated under reduced pressure. Theresultant residue was purified by silica gel column chromatography(ethyl acetate/CH₂Cl₂=1/8) to give 2.14 (782 mg, 99% from 2.11) as awhite solid. ¹H NMR (500 MHz, CDCl₃): δ 2.17 (s, 3H), 3.47 (d, 2H, J=7.0Hz), 5.04-5.07 (m, 2H), 5.85 (ddt, 1H, J=17.0, 10.0, 7.0 Hz), 6.87-6.90(m, 2H), 6.94-6.97 (m, 2H), 7.31-7.35 (m, 2H), 7.44-7.47 (m, 2H), 7.54(br.s, 1H); ¹³C NMR (75 MHz, CDCl₃): δ 24.4, 38.5, 117.5, 118.7, 119.6,121.7, 129.0, 132.9, 133.6, 133.7, 153.1, 156.7, 168.4; HRMS (FAB) calcdfor C₁₇H₁₇NO₂S (M⁺) 299.0980, found 299.0980.

{4-[4-(Methanesulfonamido)phenoxy]phenylsulfonyl}methyloxirane. To astirred solution of 2.13 (544 mg, 1.62 mmol) in CH₂Cl₂ (20 ml) was addedmCPBA (4.2 g, 17.05 mmol) at ice-water temperature, and the mixture wasstirred at room temperature for 9 days. With ice-cooling, the reactionwas quenched with saturated Na₂S₂O₃ and saturated NaHCO₃ solutions, andthe mixture was extracted with ethyl acetate (3×). The combined organiclayer was washed with saturated Na₂S₂O₃ solution, saturated NaHCO₃solution, water and brine, dried over Na₂SO₄, and concentrated underreduced pressure. The resultant residue was purified by silica gelcolumn chromatography (ethyl acetate/hexane=3/2) to give 2.2 (386 mg,62%) as a white solid. ¹H NMR (300 MHz, CDCl₃): δ 2.49 (dd, 1H, J=5.1,1.8 Hz), 2.83 (m, 1H), 3.06 (s, 3H), 3.27-3.36 (m, 3H), 6.77 (br.s, 1H),7.08-7.11 (m, 4H), 7.28-7.33 (m, 2H), 7.88-7.93 (m, 2H); ¹³C NMR (125MHz, acetone-d₆): δ 39.4, 45.9, 46.6, 59.9, 118.3, 122.3, 123.6, 131.6,134.6, 136.5, 152.7, 163.5; HRMS (FAB) calcd for C₁₆H₁₇NO₆S₂ (M⁺)383.0497, found 383.0496.

[4-(4-Nitrophenoxy)phenylsulfoyl]methyloxirane. This material wasprepared in the same manner as described for 2.2, with the exceptionthat 2.11 was used in place of 2.13. The crude material was purified bysilica gel column chromatography (ethyl acetate/hexane, 2/3) to give 2.4(56%) as a pale yellow solid. ¹H NMR (500 MHz, CDCl₃): δ 2.51 (dd, 1H,J=4.5, 2.5 Hz), 2.83 (t, 1H, J=4.5 Hz), 3.28 (dd, 1H, J=14.0, 7.0 Hz),3.35 (m, 1H), 3.41 (dd, 1H, J=14.0, 4.0 Hz), 7.16-7.17 (m, 2H),7.23-7.25 (m, 2H), 7.99-8.01 (m, 2H), 8.28-8.30 (m, 2H); ¹³C NMR (125MHz, CDCl₃): δ 45.7, 45.8, 59.6, 119.1, 119.6, 126.2, 131.0, 134.9,144.0, 160.2, 160.8; HRMS (FAB) calcd for C₁₅H₁₄NO₆S (M+H⁺) 336.0542,found 336.0545.

[4-(4-Acetamidophenoxy)phenylsulfonyl]methyloxirane. This material wasprepared in the same manner as described for 2.2, with the exceptionthat 2.14 was used in place of 2.13. The crude material was purified bysilica gel column chromatography (ethyl acetate/hexane=3/1) to give 2.6(34%) as a white semi-solid. ¹H NMR (300 MHz, CDCl₃): δ 2.20 (s, 3H),2.48 (dd, 1H, J=4.5, 1.5 Hz), 2.82 (m, 1H), 3.26-3.34 (m, 3H), 7.03-7.08(m, 4H), 7.41 (br.s, 1H), 7.55-7.58 (m, 2H), 7.85-7.88 (m, 2H); ¹³C NMR(75 MHz, CDCl₃): δ 24.1, 45.7, 45.8, 59.8, 117.6, 120.8, 122.0, 130.4,133.0, 135.4, 151.1, 163.0, 168.5; HRMS (FAB) calcd for C₁₇H₁₈NO₅S(M+H⁺) 348.0906, found 348.0913.

{4-[4-(Methanesulfonamido)phenoxy]phenylsulfonyl}methylthiirane.

To a stirred solution of 2.2 (82 mg, 0.21 mmol) in MeOH-THF (3:1, 2 ml)was added thiourea (41 mg, 0.53 mmol) at room temperature, and themixture was stirred overnight at the same temperature. Afterconcentration under reduced pressure, the residue was dissolved intoethyl acetate. The ethyl acetate solution was washed with water andbrine, dried over Na₂SO₄, and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography (ethylacetate/hexane, 1/1) to give 2.3 (67 mg, 77%) as a white solid. ¹H NMR(300 MHz, CDCl₃): δ 2.17 (dd, 1H, J=5.1, 1.8 Hz), 2.55 (dd, 1H, J=6.3,1.2 Hz), 3.06 (s, 3H), 3.07 (m, 1H), 3.22 (dd, 1H, J=14.1, 7.8 Hz), 3.50(dd, 1H, J 14.1, 5.7 Hz), 6.72 (br.s, 1H), 7.08-7.12 (m, 4H), 7.30-7.33(m, 2H), 7.87-7.91 (m, 2H); ¹³C NMR (125 MHz, acetone-d₆): δ 24.4, 27.2,39.3, 62.6, 118.4, 122.3, 123.6, 131.9, 133.9, 136.4, 152.7, 163.6; HRMS(FAB) calcd for C₁₆H₁₇NO₅S₃ (M⁺) 399.0269, found 399.0268.

[4-(4-Nitrophenoxy)phenylsulfonyl]methylthiirane. This material wasprepared in the same manner as described for 2.3, with the exceptionthat 2.4 was used in place of 2.2. The crude material was purified bysilica gel column chromatography (ethyl acetate/hexane, 1/3) to give 5(79%) as a pale yellow solid. ¹H NMR (500 MHz, CDCl₃): δ 2.20 (dd, 1H,J=5.5, 2.0 Hz), 2.58 (dd, 1H, J=6.0, 2.0 Hz), 3.10 (m, 1H), 3.31 (dd,1H, J=14.0, 7.5 Hz), 3.52 (dd, 1H, J=14.0, 6.5 Hz), 7.15-7.18 (m, 2H),7.23-7.26 (m, 2H), 7.97-8.00 (m, 2H), 8.28-8.31 (m, 2H); ¹³C NMR (125MHz, CDCl₃): □ 24.0, 26.0, 62.5, 119.1, 119.8, 126.2, 131.2, 134.4,160.3, 160.8; ¹³C NMR (125 MHz, acetone-d₆): δ 24.5, 27.2, 62.6, 120.2,121.0, 127.1, 132.3, 136.2, 145.0, 161.1, 162.4; HRMS (FAB) calcd forC₁₅H₁₄NO₅S₂ (M+H⁺) 352.0313, found 352.0297.

[4-(4-Acetamidophenoxy)phenylsulfonyl]methylthiirane. This material wasprepared in the same manner as described for 2.3, with the exceptionthat 2.6 was used in place of 2.2. The crude material was purified bysilica gel column chromatography (ethyl acetate/hexane, 3/2 to 2/1) togive 7 (76%) as a white solid. ¹H NMR (300 MHz, CDCl₃): δ 2.16 (dd, 1H,J=5.1, 1.8 Hz), 2.20 (s, 3H), 2.54 (dd, 1H, J=6.3, 1.5 Hz), 3.06 (m,1H), 3.19 (dd, 1H, J=14.1, 7.8 Hz), 3.52 (dd, 1H, J=14.1, 5.7 Hz),7.03-7.08 (m, 4H), 7.52 (br.s, 1H), 7.56-7.59 (m, 2H), 7.84-7.87 (m,2H); ¹³C NMR (125 MHz, CDCl₃): δ 24.1, 24.4, 26.0, 62.6, 117.4, 121.0,121.8, 130.7, 131.6, 135.2, 150.7, 163.1, 168.6; HRMS (FAB) calcd forC₁₇H₁₈NO₄S₂ (M+H) 364.0677, found 364.0651.

Assessment of Inhibitor Solubility—Aliquots (10 μL) of the solutions ofthe thiirane compounds 2.1, 2.3, 2.5, and 2.7 in DMSO (e.g., 10 mM, 12mM, 14 mM and higher concentrations) were added to 990 μL of buffer R[50 mM HEPES (pH 7.5), 0.15 M NaCl, 5 mM CaCl₂, 0.01% Brij-35, 1% DMSO]at 37° C. Each mixture was inspected for clarity (or turbidity) tocalculate the approximate upper limit of solubility.

Enzymatic Activity Assays—Enzymatic activity was monitored withsynthetic peptide, fluorescence-quenched substrates from PeptidesInternational, Inc. (Louisville, Ky.). The activities of MMP-2, MMP-9,MMP-7 and MMP-14 were monitored with MOCAcPLGLA₂pr(Dnp)AR-NH₂ atexcitation and emission wavelengths of 328 and 393 nm, respectively, inbuffer R MOCAcRPKPVE(Nva)WRK(Dnp)NH₂ was the fluorogenic substrate usedto measure MMP-3 at 325 and 393 nm, in buffer R. MMP-1 was assayed with(Dnp)P(Cha)GC(Me)HAK(NMa)NH₂ at 340 and 440 nm, in a buffer consistingof 50 mM Tris (pH 7.6), 200 mM NaCl, 5 mM CaCl₂, 20 mM ZnSO₄, 0.05%Brij.35. Less than 10% substrate hydrolysis was monitored (Knight, C. G.(1995) Methods Enzymol 248, 18-34).

Fluorescence was measured using a Photon Technology International (PTI)spectrofluorometer, equipped with RadioMaster™ and FeliX™ hardware andsoftware, respectively. The excitation and emission band passes were 1and 3 nm, respectively. An integration time of 4 seconds was used fordata acquisition. The assays were carried out at 25° C. and the cuvetteholder was kept at the same temperature. Quartz or disposable acrylicmicro or semi-micro cuvettes from Sarstedt (Newton, N. C.) and PerfectorScientific (Atascadero, Calif.), respectively, were used.

Enzyme Inhibition Studies—Slow-binding enzyme inhibition was monitoredcontinuously for 20-60 min, by adding the enzyme (0.5-1 nM) to asolution of buffer R containing the appropriate fluorogenic substrateand increasing concentrations of the inhibitor (final volume 2 ml) inacrylic cuvettes with stifling. The progress curves were non-linearleast squares fitted to Equation 1 (Muller-Steffner et al. (1992) J BiolChem 267, 9606-9611):F=v _(s) t+(v _(o) −v _(s))(1−exp(−kt))/k+F _(o)  (Eq. 1)where v_(o) represents the initial rate, v_(s) the steady state rate, kthe apparent first-order rate constant characterizing the formation ofthe steady-state enzyme-inhibitor complex, and F_(o) the initialfluorescence, using the program Scientist (MicroMath ScientificSoftware, Salt Lake City, Utah). Association and dissociation rateconstants (k_(on) and k_(off), respectively) were obtained from theslope and intercept, respectively, of plots of the apparent first-orderrate constant k versus the inhibitor concentration according to Equation2:k=k _(off) +k _(on) [I]/(1+[S]/K _(m))  (Eq. 2)describing a one-step association mechanism (Scheme 3),

where S is the fluorogenic peptide substrate used and the EI* is theproduct of slow-binding inhibition. The expression for k_(m), includesthe requisite conformational change necessary for the formation of EI*.The K_(m) values used for the reaction of MMP-2, MMP-9 and MMP-14 withthe fluorogenic substrate MOCAcPLGLA₂pr(Dnp)AR-NH₂ were 2.46±0.34,3.06±0.74 (Olson, M. W., Gervasi, D. C., Mobashery, S., and Fridman, R.(1997) J Biol Chem 272, 29975-29983) and 6.9±0.6 μM (Toth, M., Bernardo,M. M., Gervasi, D. C., Soloway, P. D., Wang, Z., Bigg, H. F., Overall,C. M., DeClerck, Y. A., Tschesche, H., Chem., M. L., Brown, S.,Mobashery, S., and Fridman, R. (2000) J Biol Chem 275, 41415-41423),respectively. The inhibition constant, K_(i), was given byk_(off)/k_(on). Alternatively, K_(i) values were obtained by plotting(v_(o)−v_(s))/v_(s) versus the inhibitor concentration, according toEquation 3:(v _(o) −v _(s))/v _(s) =[I](K _(i)(1+[S]/K _(m)))  (Eq. 3).

For analysis of simple linear competitive inhibition, reaction mixturescontaining the enzyme (˜1 nM) and increasing concentrations of theinhibitor, in buffer R (final volume 1 mL), were incubated for ˜16hours, at 25° C. in semi-micro acrylic cuvettes. The remaining enzymaticactivity was measured with the appropriate synthetic peptide fluorogenicsubstrate for 5-10 minutes. The initial velocities for the reaction ofthe enzyme with the substrate were determined by linear regressionanalysis of the fluorescence versus time traces using FeliX™. Theseinitial rates were fitted to Equation 4 (Segel, I. H. Enzyme Kinetics,John Wiley & Sons, Inc.: New York (1975)):v _(i) /v _(o)=(K _(m) +[S])/(K _(m)(1+[I]/K _(i))+[S]))  (Eq. 4)where v_(i) and v_(o) represent the initial velocity in the presence andabsence of inhibitor, respectively, using the program Scientist.

Equilibrium Dialysis—Mixtures of enzyme (10 nM) in the presence andabsence of inhibitor (1 mM) were incubated at room temperature for ˜3hours. The remaining enzyme activity was measured with the appropriatefluorogenic substrate, as described above. Part of the reaction mixture(˜150 μL) was dialyzed in either dialysis tubing (Invitrogen) or in a0.1-0.5 mL capacity Slide-A-Lyzer® dialysis cassette (Pierce), againstbuffer R (3×1 L) containing no DMSO, at room temperature, for >4 hourperiods prior to change of buffer to allow for equilibration, over a 48hour period. The remaining of the inhibition mixture was left on arotator, at room temperature, over the same period of time. Both thedialyzed and non-dialyzed solutions were tested for MMP activity usingthe proper fluorogenic substrate. Enzyme activity was expressed inpercentage relative to that in the absence of inhibitor.

Cell Culture—Human HeLa S3 cells were obtained from the American TypeCulture Collection (ATTC, Manassas, Va.) (CCL-2.2) and grown insuspension in MEM Spinner (Quality Biologicals, Inc., Gaithersburg, Md.)supplemented with 5% horse serum. Nonmalignant, monkey kidney epithelialcells, BS-C-1 (CCL-26), and human fibrosarcoma cells, HT1080 (CCL-2.2),were obtained from ATCC and cultured in Dulbecco's modified Eagle'smedium (DMEM, Invitrogen, Carlsbad, Calif.), supplemented with 10% fetalbovine serum (FBS) and antibiotics.

Recombinant Vaccinia Viruses—Recombinant vaccinia viruses encoding forT7 RNA polymerase (vTF7-3) or pro-MMP-2 (vT7-72), pro-MMP-9 (vT7-92),MT1-MMP (vTF-MT1), TIMP-1 (vTF-TEVT-1) and TIMP-2 (vSC59-T2) wereproduced by homologous recombination as previously described (Fuerst, T.R., Earl, P. L., and Moss, B. (1987) Mol Cell Biol 7, 2538-2544).

Recomibinant Proteins, Enzymes and Inhibitors—Human recombinantpro-MMP-2 and pro-MMP-9, TIMP-1 and TIMP-2 were expressed in HeLa S3cells infected with the corresponding recombinant vaccinia viruses andpurified to homogeneity from the media as previously described (Olson,M. W., Gervasi, D. C., Mobashery, S., and Fridman, R. (1997) J Biol Chem272, 29975-29983). Pro-MMP-2 and pro-MMP-9 were activated by incubationwith 1 mM p-aminophenylmercuric acetate (APMA) for ˜2 hours at 37° C. aspreviously described (Olson, M. W., Gervasi, D. C., Mobashery, S., andFridman, R. (1997) J Biol Chem 272, 29975-29983). APMA was dialyzed outin collagenase buffer (50 mM Tris-HCl (pH 7.5) 5 mM CaCl₂, 150 mM NaCland 0.02% Brij-35).

Human recombinant active MMP-1, and MMP-7 were from R&D Systems(Minneapolis, Minn.) and Chemicon International (Temecula, Calif.),respectively, and the recombinant catalytic domains of human MMP-3 andMMP-14 were from CalBiochem (San Diego, Calif.). Active enzymeconcentration was determined by active-site titration with solutions ofeither TIMP-1 or TIMP-2 with known concentration. The hydroxamate-basedMMP inhibitor BB-94 was synthesized in the Mobashery laboratory andGM6001 (hydroxamate inhibitor) was purchased from Chemicon. Stocksolutions of BB-94, GM6001, and compounds 2.2-2.7 were prepared in DMSOin the mM concentration range.

Pro-MMP-2 Activation on Cells—Confluent BS-C-1 cells in 12-well plates,were co-infected with v-TF7-3 and vTF-MT1 viruses for 45 min, ininfection medium (DMEM supplemented with 2.5% PBS and antibiotics), at37° C., as described by Hernandez-Barrantes et al. ((2000) J Biol Chem275, 12080-12089). The infection medium was removed and the cells wereincubated overnight with serum-free DMEM supplemented with L-glutamineand antibiotics containing increasing concentrations (0-5 μM) of thesynthetic MMP inhibitors (MMPIs). The cells were washed twice withphosphate buffer saline (PBS), and incubated for 6 hours with serum-freeDMEM containing pro-MMP-2 (10 nM). The cells were rinsed twice with coldPBS and lysed with cold lysis buffer (25 mM Tris-HCl (pH 7.5), 1% IGEPALCA-630, 100 mM NaCl) containing protease inhibitors (1 pellet ofComplete Mini, EDTA-free protease inhibitor mixture from RocheDiagnostics/10 mL of buffer). The lysates were then subjected to gelatinzymography to monitor pro-MMP-2 activation and to immunoblot analysis todetect MT1-MMP expression and processing.

Gelatin Zymography and Immunoblot Analysis—Gelatin zymography wasperformed using 10% Tris-glycine SDS-polyacrylamide gels, containing0.1% gelatin, as previously described (Toth, M., Gervasi, D. C., andFridman, R. (1997) Cancer Res. 57, 3159-3167). The samples forimmunoblot analysis were subjected to reducing SDS-PAGE followed bytransfer to nitrocellulose membranes. MT1-MMP was probed with rabbitpolyclonal antibody 815 to MT1-MMP, from Chemicon.

Migration and Invasion Assays—For migration assays, HT1080 cells werecultured in 6-well plates in complete media until they reachedconfluence. Prior to the migration assay, the cells were treated withserum-free media containing mitomycin C (25 μg/mL), in the presence andabsence of concanavalin A (ConA) (20 μg/mL, 30 minutes) to inducepro-MMP-2 activation (Gervasi, D. C., Raz, A., Dehem, M., Yang, M.,Kurkinen, M., and Fridman, R. (1996) Biochem Biophys Res Commun 228,530-538). Scratch wounds were then carefully made in the confluentmonolayer using a disposable plastic pipette tip. After gentle rinsingtwice with PBS to remove detached cells, serum-free media containingincreasing concentrations of inhibitor 2.3 were added, and the cellswere incubated at 37° C. for various times. Photographs were taken usingan Olympus Model DF 12-2 camera connected to a Nikon TMS-F microscope at10× magnification, at the indicated time points. The extent of woundclosure in the presence or absence of inhibitor was quantified bymeasuring the width of the wound with a ruler using an amplifiedPowerPoint figure.

Tumor cell invasion was carried out in 8-μm pore Transwell inserts(Becton Dickinson, Boston, Mass.) coated with 50 μg Matrigel per filter.HT1080 cells suspended in serum-free DMEM containing 0.1% bovine serumalbumin (BSA) and various doses of inhibitor 2.3 (0.1-10 μM) or 1% DMSO(vehicle) were seeded (2×10⁵ cells/insert) on the Matrigel-coatedinserts. The lower compartment was filled with DMEM containing 5% FBS.After an 18 h-incubation at 37° C. in a humidified atmosphere with 5%CO₂, the upper surface of the membrane in each insert was wiped off witha cotton swab to remove all of the non invading cells. The cells thatmigrated to the lower side of the Matrigel-coated filter were fixed andstained with Diff-Quik® (Dade Behring Inc., Newark, Del.), and countedunder a microscope in three different fields. Each treatment was assayedin quadruplicate.

Chemosensitivity Assay—Cell viability after exposure of the cells to theinhibitors was assessed by2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,monosodium salt (WST-1, Roche Diagnostics Gmbh, Mannheim, Germany)staining. Briefly, HT1080 cells (2×10⁴ cells/well) were seeded in96-well culture plates in complete medium. After overnight culture, themedium was replaced with serum and phenol-free media containing 0.1% BSAand supplemented with or without inhibitor 2.3 (0-10 μM finalconcentrations). Control medium was supplemented with 1% DMSO. After 18hours incubation, WST-1 (10 μL/well) was added and the difference inabsorbance at 450 and 655 (reference filter) nm was measured using aBio-Rad Benchmark microplate reader. Data were collected using theMicroplate Manager software. The absorbance of blank wells containingcontrol media but no cells (typically <5%) was subtracted. Eachtreatment was assayed in quadruplicate.

Results And Discussion:

Design and synthesis of MMP inhibitors—The computational model forbinding of inhibitor 2.1 to the active site of M-2 is shown in FIG. 3B.The site for substitution at the para position of the terminal phenylring of the inhibitor is indicated by an arrow. Scheme 4 illustrates thesynthetic route to oxiranes 2.2, 2.4 and 2.6, and thiiranes 2.3, 2.5 and2.7.

Chemoselective allylation of commercially available 4-hydroxythiophenol(2.8) provided phenol 2.9 (Goux et al. Tetrahedron 1994, 50,10321-10330) in 70% yield, which was subsequently coupled with1-fluoro-4-nitrobenzene (2.10) in the presence of cesium carbonate toafford the diphenyl ether 2.11 in 89% yield. The nitro group of 2.11 wasreduced over elemental zinc and the resulting amine 2.12 was treatedwith methanesulfonyl chloride or acetic anhydride to give thecorresponding amides 2.13 and 2.14, respectively, in high yields.Oxidation of 2.13, 2.11 and 2.14 to their corresponding oxiranes 2.2,2.4 and 2.6 required excess of mCPBA (10-12 equiv.) and took longreaction time (9-10 days) due to the low reactivity of the olefinmoieties; the isolated yields of the oxiranes were moderate (34-62%).

Finally, the conversion of compounds 2.2, 2.4 and 2.6 to thecorresponding thiiranes 2.3, 2.5 and 2.7 was accomplished by thetreatment with thiourea in good yields. Compounds 2.3 and 2.7 hadimproved solubility in aqueous solution compared to the prototypicinhibitor 2.1. Solubility was investigated in 50 mM HEPES (pH 7.5), 0.15M NaCl, 5 mM CaCl₂, 0.01% Brij-35, 1% DMSO, which was the buffer that weused for all the kinetic experiments (see below). The maximal solubilityfor the samples were 2.1: 80 μM; 2.3: 340 μM; 2.5: 60 μM; 2.7: 540 μM.

Enzyme inhibition kinetics—The mechanism of action of this class of MMPinhibitors stipulates that the thiirane sulfur would coordinate with theactive-site zinc ion (FIG. 2). Consistent with this expectation, thethree synthetic thiiranes of this study (compounds 2.3, 2.5 and 2.7) areexcellent inhibitors of gelatinases (and of MT1-MMP in the case ofinhibitor 2.3) (Table 2), whereas the corresponding oxiranes (compounds2.2, 2.4 and 2.6) are either poor inhibitors or not inhibitory at alltoward all the tested MMPs (Table 3). For Table 2, the enzymes (0.5-1nM) were added to a solution of the proper synthetic fluorogenicsubstrate and increasing concentrations of the inhibitor in buffer R.Substrate hydrolysis was monitored for up to 30 minutes. The kineticparameters were evaluated as described above. For Table 2, the enzymes(0.5-1 nM) were incubated with increasing concentrations of inhibitor inbuffer R. The remaining activity was measured with the appropriatesynthetic fluorogenic substrate. The kinetic parameters for rapid,competitive inhibition were evaluated as described above.

TABLE 2 Kinetic parameters for MMP inhibition by compounds 2.3, 2.5, and2.7 k_(on) k_(off) K_(i) Enzyme M⁻¹s⁻¹ s⁻¹ μM Compound 2.3 MMP-2 (2.1 ±0.5) × 10⁴ (3.5 ± 1.6) × 10⁻⁴ 0.016 ± 0.09  MMP-9 (4.9 ± 0.2) × 10³ (9.0± 2.0) × 10⁻⁴ 0.18 ± 0.05 MMP-14 (6.9 ± 0.8) × 10² (6.4 ± 0.5) × 10⁻⁴0.9 ± 0.1 MMP-7 295 ± 10  MMP-3 3.6 ± 0.2 MMP-1 NI^(a) up to 25 μMCompound 2.5 MMP-2 (1.9 ± 0.6) × 10³ (1.3 ± 0.2) × 10⁻³ 0.7 ± 0.2 MMP-91.0 ± 0.1 MMP-14 4.9 ± 0.3 MMP-7 153 ± 16  MMP-3 131 ± 9  MMP-1 67 ± 18Compound 2.7 MMP-2 (1.2 ± 0.3) × 10⁴ (1.3 ± 0.3) × 10⁻³ 0.11 ± 0.04MMP-9 0.13 ± 0.01 MMP-14 0.68 ± 0.05 MMP-7 39 ± 3  MMP-3 12.2 ± 0.9 MMP-1 5.4 ± 0.4 ^(a)NI: not inhibiting

TABLE 3 Kinetic parameters for MMP inhibition by compounds 2.2, 2.4, and2.6 K_(i) Enzyme μM Compound 2.2 MMP-2  2.4 ± 0.5 MMP-9 NI^(a) up to 26μM MMP-14 NI up to 170 μM MMP-7 379 ± 29 MMP-3 NI up to 170 μM MMP-1 45± 9 Compound 2.4 MMP-2 13 ± 1 MMP-9 25 ± 5 MMP-14  76 ± 13 MMP-7 130 ±15 MMP-3 NI up to 190 μM MMP-1 NI up to 298 μM Compound 2.6 MMP-2  0.84± 0.03 MMP-9 34 ± 3 MMP-14 NI up to 230 μM MMP-7 NI up to 184 μM MMP-3NI up to 188 μM MMP-1 NI up to 154 μM ^(a)NI: not inhibiting

In slow-binding inhibition, on binding of the inhibitor to the enzyme,the complex undergoes a requisite conformational change that is notreadily predisposed for the reversal of the inhibition (Duggleby et al.(1982) Biochemistry 21, 3364-3370; Morrison et al. Adv. Enzymol. Relat.Areas Mol. Biol. 61, 201-301; and Szedlacsek and Duggleby (1995) MethodsEnzymol. 249, 144-180). The slow-binding inhibitor shows a uniqueprofile for the onset of inhibition that is discerned by non-linearprogress curves. Slow-binding behavior was seen for inhibitor 2.3 (withMMP-2, MMP-9, and MMP-14), for inhibitor 2.5 (only with MMP-2), and forinhibitor 2.7 (only with MMP-2) (FIG. 4).

The second-order rate constants for the onset of slow-binding inhibition(k_(on)) are rapid (10² to 10⁴ M⁻¹s⁻¹) and the first-order rateconstants for the reversal of the process from the non-covalentenzyme-inhibitor complexes (k_(off)) are slow (10⁻⁴ to 10-35-1; e.g.,the t_(1/2) for reversal for inhibitor 2.3 with MMP-2 and MMP-9 are 34minutes and 13 minutes, respectively). The dissociation constants forthe non-covalent complexes (K_(i)) that result from slow-bindinginhibition are computed from the ratios of k_(off)/k_(on). Compounds2.3, 2.5, and 2.7 are clearly selective for gelatinases, with 2.3showing the slow-binding behavior with MMP-14 as well. The K_(i) valuesof the slow-binding component for inhibition by 2.3 (16±9 nM, 180±50 nM,0.9±0.1 μM for MMP-2, MMP-9 and MMP-14, respectively), 2.5 (700±200 nMfor MMP-2), and 2.7 (110±40 nM for MMP-2) are listed in Table 2.

It is noteworthy that the inhibition profiles for inhibitors 2.1, 2.3,2.5, and 2.7 as mechanism-based inhibitors are different from oneanother, despite the similar structural template for the class. Briefly,inhibitor 2.1 can inhibit both MMP-2 and MMP-9 (the gelatinases),inhibitor 2.3 inhibits the gelatinases plus MMP-14, and mostinterestingly, inhibitors 2.5 and 2.7 are mechanism-based inhibitorsonly for MMP-2. Furthermore, these are nanomolar inhibitors for theirtargeted enzymes and exhibit comparable values for the k_(on) andk_(off) parameters for the slow-binding components of their kinetics.

Covalent vs. non-covalent inhibition of MMPs—The thiirane class of MMPinhibitors was designed to be covalent enzyme inhibitors. On formationof the non-covalent enzyme-inhibitor complex, the ubiquitous active siteglutamates of MMPs (Glu⁴⁰⁴ for MMP-2, for example) were expected to becovalently modified by the inhibitor with the requisite thiirane ringopening (FIG. 2). The kinetics of inhibition indicate two components, anon-covalent stage (slow-binding) and a subsequent stage that may beattributed to the covalent modification of the active site glutamate, aswill be outlined.

The covalent component of inhibition results in modification of theglutamate as an ester on its side chain carboxylate. The earlier X-rayabsorption spectroscopy analysis with inhibitor 2.1 (Kleifeld et al.(2001) J Biol Chem 276, 17125-17131) had provided evidence for thecovalent bond formation, in that on the onset of inhibition the methodrevealed the formation of a thiolate from the thiirane of the inhibitor(ring opening), coordinated to the active site zinc ion.

Whereas a slow-binding step need not necessarily be a prerequisite forcovalent chemistry, both the mechanism-based process leading to covalentenzyme modification and the slow-binding behavior producetime-dependence for the loss of activity seen with these inhibitors(FIG. 4). Our experience with inhibitor 2.1 had shown that slow-bindingled to covalent chemistry, with a longevity for the final inhibitedspecies substantially exceeding the duration that would have beenanticipated from four times the t_(1/2) for recovery of activity fromthe slow-binding component of inhibition (in other words, fourhalf-lives leading to an anticipated 94% recovered of activity due tothe non-covalent component). This is the case with inhibitors 2.3, 2.5,and 2.7 as well.

The slowest t_(1/2) calculated for recovery of activity from thenon-covalent slow-binding species for the best inhibition (compound 2.3with MMP-2) is 34 minutes. Yet, a mere 1% of activity recovery was seenfor MMP-2 inhibited by inhibitor 2.3 after 48 hours of dialysis. Fourhalf-lives for recovery from inhibition (94% anticipated recoveredactivity) with this inhibitor and MMP-2 should be achieved in just under2.5 hours (136 minutes), were it merely the slow-binding event thataccounted for MMP-2 inhibition. This is clearly not the case and theinhibited enzyme is more stable than the k_(off) (from which t_(1/2) isevaluated) indicates. The results of dialyses for inhibitors 2.3, 2.5,and 2.7 are given in FIG. 5.

Having documented above that mere slow-binding behavior cannot beresponsible for the observed complete inhibition, an explanation wassought as to why any recovery of activity should be seen, if covalentchemistry is involved. The answer is that stability of covalent bonds isrelative. Esters are among the least stable covalent bonds in aqueoussolution (Westheimer, F. H. (1987) Science 235, 1173-1178). This bondwould undergo hydrolysis, resulting in recovery of activity. The processaccelerates when there is a more significant exposure of the ester bondto water, conditions that can arise when the protein is denatured.

Matrix-assisted laser desorption time-of-flight mass spectrometry(MALDI-TOF MS) analysis, performed on an Applied Biosystems Voyager-DESTR (Framingham, Mass.) at the Harvard Microchemistry and ProteomicsAnalysis Facility (Cambridge, Mass.), was attempted on samplescontaining MMP-2 (10 μM) in the presence and absence of inhibitor 2.3 todetect a shift in molecular mass consistent with a complex of activeMMP-2 (˜62 kDa) with the inhibitor. However, after several attempts withdifferent conditions we failed to detect a 400 Da addition in molecularmass to the 62-kDa peak. The difficulty is that at this high end of massdetection the signals are broadened and the identification of the smallincremental increase due to the mass of the inhibitor was not possiblewithin the resolution of the instrument.

Effect of Gelatinase Inhibitors on pro-MMP-2 Activation byMT1-MMP—MT1-MMP has been identified as the physiological activator ofpro-MMP-2 (Strongin et al. (1993) J Biol Chem 268, 14033-14039). Thisreaction is regulated at multiple levels and its rate is significantlyenhanced by TOIP-2, which, by binding active MT1-MMP, acts as a“receptor” for pro-MMP-2 on the cell surface (Westheimer, F. H. (1987)Science 235, 1173-1178). The binding of pro-MMP-2 to the MT1-MMP/TIMP-2complex, facilitates the first pro-MMP-2 cleavage by a neighboringTIMP-2-free MT1-MMP molecule (Strongin et al. (1995) J Biol Chem 270,5331-5338). Pro-MMP-2 activation requires a second autolytic cleavage(Will et al. (1996) J Biol Chem 271, 17119-17123), leading to fullactivation.

It has been shown previously that broad-spectrum synthetic MMPinhibitors, e.g. marimastat, enhance pro-MMP-2 activation by MT1-MMP inthe presence of TIMP-2 (Toth et al. (2000) J Biol Chem 275,41415-41423), a process that appears to involve stabilization of matureMT1-MMP at the cell surface by the MMP inhibitor. This enhancing effecton pro-MMP-2 activation was not observed when the cells were exposed toinhibitor 2.1, which exhibits lower affinity towards MT1-MMP, a featureof its selectivity for inhibition of gelatinases.

Therefore, it was proposed that non-specific targeting of MT1-MMP bybroad-spectrum MMP inhibitors might, under certain conditions, elicit acounterproductive effect by enhancing the activity of theMT1-MMP/gelatinase A axis (Bernardo, M. M., Brown, S., Li, Z. H.,Fridman, R., and Mobashery, S. (2002) J Biol Chem 277, 11201-11207).Because inhibitor 2.3 is also selective for the gelatinases, it waspostulated that it might behave like inhibitor 2.1 in a cellular systemof pro-MMP-2 activation by MT1-MMP in the presence of TIMP-2. To thisend, BS-C-1 cells, which express low levels of endogenous TIMP-2, wereinfected to express MT1-MMP, and incubated with pro-MMP-2 in thepresence of either GM6001, a broad-spectrum MMP inhibitor or inhibitor2.3, as described by Toth and co-workers ((2000) J Biol Chem 275,41415-41423). Pro-MMP-2 activation was followed by gelatin zymography.As shown in FIG. 6 a, exposure of the MT1-MMP-expressing cells to aslittle as 40 nM GM6001, induced pro-MMP-2 activation, as determined bythe appearance of the active form. Higher inhibitor concentrationsfurther enhanced pro-MMP-2 activation, under these conditions.

Of note, this enhancing effect of broad-spectrum MMP inhibitors such asGM6001 requires the endogenous TIMP-2, as have shown previously shown byToth and co-workers ((2000) J Biol Chem 275, 41415-41423). Consistently,GM6001 caused a dose-dependent accumulation of active MT1-MMP (57 kDa)(FIG. 6B). In contrast, when the cells were incubated with inhibitor 2.3(up to 4 μM), pro-MMP-2 activation was not observed. Also, theaccumulation of active MT1-MMP was not observed with inhibitor 2.3,consistent with its reduced affinity for this protease when compared toMMP-2 (Table 2).

Although inhibitor 2.3 is also a mechanism-based inhibitor for MT1-MMP,its lower affinity relative to MMP-2 is likely to preclude thisinhibitor to influence pro-MMP-2 activation under these conditions. Itis also possible that covalent inhibition of MT1-MMP, as opposed to areversible inhibition, alters the availability of the active site ofMT1-MMP for TIMP-2 binding, a prerequisite for pro-MMP-2 activation((2000) J Biol Chem 275, 41415-41423). Although more studies arerequired, these results suggest that the concept behind inhibitor 2.3 isa promising framework from which to further develop more effective andselective MT1-MMP inhibitors, a key protease in tumor cell invasion.Nevertheless, these studies further demonstrate the selectivity ofinhibitor 2.3 in a live cellular system and lend credit to thehypothesis that selectivity, rather than affinity, may be key to thesuccessful therapeutic application of synthetic MMP inhibitors.

Inhibitor 2.3 inhibits HT1080 cell migration and invasion—It is wellestablished that tumor cell migration and invasion depend on gelatinaseactivity. Therefore, we wished to evaluate the effect of inhibitor 2.3,which is selective for the gelatinases, on the migration and invasion ofHT1080 cells, as described above. Cell migration was monitored underconditions of pro-MMP-2 activation, which was achieved by ConAtreatment, and inhibition of cell proliferation. As shown in FIGS. 7Aand B, exposure of HT1080 cells to various doses (0-20 μM) of inhibitor2.3 significantly inhibited (80% at 2 μM) their migration in a scratchwound assay when compared to untreated cells. Likewise, the ability ofHT1080 cells to invade Matrigel-coated filters was significantly reducedby inhibitor 2.3 and as little as 100 nM of inhibitor caused >25%inhibition of HT1080 cell invasion (FIG. 7C).

These effects of inhibitor 2.3 could not be ascribed to cytotoxicity asno evidence of cell toxicity was detected when HT1080 cells were exposedto inhibitor 2.3 up to concentrations of 10 μM, as determined using theWST-1 chemosensitivity assay (data not shown). Given the highselectivity exhibited by this compound towards MMP-2 relative to otherMMPs (Table 2), the slower migration in the presence of 200 nM ofinhibitor 2.3, a concentration too low to inhibit other MMs includingMT1-MMP, suggests that the observed effect was most likely due to MMP-2inhibition. These results further demonstrate the ability of inhibitor2.3 to act as a selective gelatinase inhibitor in cellular systems andto alter MMP-dependent processes. The new characteristics of inhibitor2.3 and its high selectivity make this inhibitor an excellent candidatefor future in vivo testing in relevant human disease models in mice.

The thiirane class of mechanism-based inhibitors was conceived, designedand prepared by us for the first time in our pursuit of selectivity ininhibition of MMPs of importance to several disease processes. We haverevealed in the present report that inhibitor 2.3 targets MMP-2, -9, and-14, whereas inhibitors 2.5 and 2.7 are inhibitory only toward MMP-2.The activities for these new inhibitors provide a unique opportunity ininvestigations of the roles of these MMPs in various disease processes.

Example 3 Synthetic Approach to Aromatic Sulfonylmethylthiiranes

A general strategy for the synthesis of aromatic sulfonylmethylthiiranesand their derivatives is described below (Scheme 5-9). The compoundsexplicitly depicted in the schemes of Example 3 have been synthesizedand their compound data is provided in the Experimental Sectionfollowing Scheme 29.

A suitable synthetic sequence to affordphenoxyphenylsulfonylmethyl-thiirane 5 is outlined in Scheme 5 (see Leeet al. Org. Lett. 2005, 7, 4463-4465; Lim, Brown, and Mobashery, J. Org.Chem. 2004, 69, 3572-3573). Synthesis commenced with a commerciallyavailable bromide, which was lithiated with n-BuLi, was then convertedto the thiolate. The thiolate was alkylated with epichlorohydrin toresult in compound 2. The hydrochloride moiety in compound 2 wastransformed to an epoxide ring, followed by sulfide oxidation by use ofm-CPBA. The resulting epoxide 4 was converted to thiirane 5 usingthiourea. When ortho- or meta-bromide are used instead of para bromide1, corresponding sulfonylmethylthiiranes 7 or 9 can be obtained by thesame sequence illustrated in Scheme 5.

The thiolate, which was generated by treatment of n-BuLi and sulfur, isa very useful intermediate because it can be reacted with otherelectrophiles, such as allylbromide and acetyl chloride, yieldingcompounds 10 and 11 (see Schemes 6 and 7), which can be ultimatelyconverted to epoxide 4, a precursor of thiirane 5 (Scheme 6).Allylthio-4-phenoxybenzene derivative 10 can be converted directly tosulfonylmethyloxirane 4 by m-CPBA. While this method cannot be appliedin some cases, dihydroxylation (J. Am. Chem. Soc. 1997, 119, 311-325)serves as an alternative route to sulfonylmethyloxirane 4. Diols 12 and13 can be converted to the epoxide either via tosylation (Heterocycles1990, 31, 1555-1562) or Mitsunobu reaction (Synthesis 1983, 116-117).The sulfide can be oxidized to the sulfone before or after formation ofthe epoxide. The second alkylation product thioacetate 11 can directlygive 3 by treatment with epichlorohydrin. When allyl bromide or glycidolare reacted with compound 11, compounds 10 and 12 can be obtained.

Aromatic amines or nitro compounds can be used to prepare compound 11 bythe route in Scheme 7. Nitro compound 13 was reduced in the presence ofPd/C and H₂, or SnCl₂ or Zn in the presence of AcOH, to provide amine14. Aromatic amine 14 was then diazotized using isoamyl nitrite andreacted with potassium thioacetate to yield aromatic thioacetate 11(Synthesis 2003, 1225-1230). The resulting thioacetate 11 was thenconverted to compound 5, by the route described in Scheme 6.

When aromatic bromide or amine are not commercially available, phenolicethers can be prepares by several methods, as illustrated in Scheme 8.

Phenolic ethers can be constructed by copper catalyzed reactions. Onemethod is by Ullmann condensation between aromatic halides and phenol.Another method is by using aromatic boronic acid and phenol.

Phenoxyphenyl bromide was constructed by a N,N-dimethylglycine promotedUllmann coupling reaction (Org. Lett. 2003, 5, 3799-3802) between phenol16 and dibromobenzene 17 in the presence of copper (I) iodide. Whensubstituted thiophenols are used, we can make thiol incorporated phenolether, which obviates the step to introduce sulfur atom to the molecule.When allylthiophenol 19 is reacted with aromatic halide 18, compound 10can be made. When dimethyl-thiocarbamic acid S-(4-phenoxyphenyl) ester20 is reacted with aromatic halide 18, compound 21 can be formed, whichwas then hydrolyzed to give free thiol under basic condition and thenalkylated with epichlorohydrin in the presence of potassium carbonate.

Phenoxyphenyl ring can be formed by the reaction of aromatic boronicacid 22, which was reacted with phenol 23 in the presence of copperacetate. Copper-mediated arylation of phenol using boronic acid waspreviously reported by Chan and Evans (Tetrahedron Lett. 1998, 39,2933-2936; and Tetrahedron Lett. 1998, 39, 2937-2940, respectively).

Some activated halides such as compounds 24-26 do not need coppercatalyst. Compounds 24-26 were smoothly transformed to phenol ether inthe presence of base such as cesium carbonate, potassium carbonate orsodium hydroxide (Scheme 9).

Compounds with functional groups such as amino (30) and hydroxyl (33)groups are useful because they can easily react with many differentelectrophiles, such as alkylbromides, acid chlorides, and sulfonylchlorides (Scheme 10). Carboxylic acid 36 is also useful in this sensefor its reactivity with alcohols or amines to afford esters 37 or amidecompounds 38.

p-Aminophenylphenoxy derivatives were synthesized according to Scheme11. 4-amino group was introduced by use of nitrobenzene fluoride 39.This activated substrate was reacted with allylthiophenol 19 in thepresence of cesium carbonate to yield1-allylthio-4-(4-nitrophenyl)benzene 40, which was then reduced to aminein the presence of zinc. Compound 42 was reacted with severalelectrophiles, followed by m-CPBA oxidation and thiirane formationreaction to afford compound 42.

Schemes 12-14 cover the synthesis of o-, m-, p-hydroxy andp-hydroxymethyl substituted phenoxyphenyl thiirane derivatives (55, 61,33). Different approaches were used to make o-, m-, and p-hydroxylsubstituted compounds. The ortho derivative was constructed by thereaction of 2-fluorobenzaldehyde and 4-bromophenol to give compound 47,then converted to 48 by three steps (Scheme 12) (Synthesis 1995, 28-30).The p-hydroxy phenoxybenzene scaffold is incorporated by using4-hydroxyphenoxy-phenybromide 49, which was converted to compound 50 viaacetylation and followed by selective bromination (J. Med. Chem. 1998,41, 1540-1554). Compound 50 was then deacetylated and protected to givecompound 51. The rest of functional group transformations were followedas in Scheme 5, except for the selective deprotection of benzyl ethergroup of compound 53. The benzyl ether was deprotected with Pd(OH)₂ inethyl acetate and i-PrOH to give compound 54.

Meta Substituted scaffold was constructed by Ullmann condensationbetween 3-benzyloxyiodobenzene and compound 20 to afford compound 57(Scheme 13) (see Org. Lett. 2003, 5, 3799-3802). Compound 57 was thenhydrolyzed to give free thiol 58 and then alkylated with epichlorohydrinin the presence of potassium carbonate. Functional group transformationfrom compound 58 to compound 61 was followed in Scheme 6.

Hydroxymethyl group was introduced by Suzuki type reaction betweenhydroxyphenyl boronic acid 62 and compound 19 (Scheme 14) (TetrahedronLett. 1998, 39, 2933-2936; Tetrahedron Lett. 1998, 39, 2937-2940). Theresulting compound 63 was converted to compound 33 by the same route inScheme 5.

A carboxymethyl group was incorporated by Ullmann reaction to result incompound 66 (Scheme 15) (Org. Lett. 2003, 5, 3799-3802). The conversionof compound 66 to 67 was accomplished according to Scheme 6 whichinvolves m-CPBA oxidation and thiirane formation using thiourea. Methylester in compound 67 was hydrolyzed to carboxylic acid 36 via formationof tributyltin ester intermediate by the treatment ofdi(tributyltin)oxide and C-18 silica gel.

The possibility of introducing different bridges between two benzenerings instead of an oxygen bridge was investigated (Scheme 16). Forthese compounds, aromatic amines 68, 71, and 74 served as the initialstarting materials, which were diazotized and then converted to thecorresponding thioesters (69, 72, 75) (see Synthesis 1998, 1171-1175 andSynthesis 2003, 1225-1230). The remainder of the synthetic route fromthe thioacetate to sulfonylmethylthiirane was followed as in Scheme 6.

When 4-thiiranylmethanesulfonylphenol 78 was reacted with alkylhalides,acid chlorides, or sulfonyl chlorides, different bridged molecules suchas compounds 79-81 were obtained (Scheme 17).

Aromatic sulfonyl thiirane other than phenoxyphenyl scaffolds were alsoprepared, as illustrated in Schemes 18-20. 4-Biphenyhnagnesium bromide82 was reacted with epichlorohydrin in the presence of CuBr-DMS to givecompound 83 (Scheme 18). The resulting hydroxychloride 83 was convertedto epoxide 84, which was reacted with vinylmagnesium bromide. Secondaryalcohol 85 was converted to a mesylate. The mesylate was then displacedplaced by thioacetate, which was hydrolyzed and alkylated to affordthiol ether 87. The conversion of the alkene moiety of 87 to thiirane 88was same as outlined in Scheme 6, which involves oxidation and thiiranering formation.

When phenoxyphenyl magnesiumbromide 90 is used instead ofbiphenylmagnesium bromide 82, compound 96 can be obtained (Scheme 19).The remainder of synthetic route is similar to that of Scheme 14.

1,3,5-Substituted benzene was chosen since it contains three positionsto be functionalized (Scheme 20). Synthesis commences from brominationof 2-amino-5-nitrophenol 97 using N-bromosuccinimide (J. Am. Chem. Soc.1997, 119, 311-325). Deamination of compound 98 afforded construction of1,3,5-substituted benzene scaffold. The resulting phenol 99 wasalkylated with halides. Suzuki coupling of compound 100 with aromaticboronic acid 18 in the presence of tetrakis(triphenylphosphine)palladiumcatalyst resulted in biphenyl derivative 101 (J. Med. Chem. 1997, 40,437-448). Nitro group in compound 101 was reduced to corresponding amine102 by catalytic hydrogenation. Conversion of thioacetate 103 to 104followed the method as described for Scheme 6.

Sulfonamide and phosphoamide groups were introduced instead of sulfonylgroup (Scheme 21-24). Sulfonamide was formed by the reaction ofphenoxyaniline 14 and allylsulfonyl chloride to yield compound 105(Scheme 21). After alkylation of amine in 105, double bond was convertedto diol 106, which was converted to sulfonylmethylthiirane 107 by thesimilar method outlined in Scheme 6.

A phosphonamide can be a good surrogate of sulfonamide because theyshare the same tetrahedral geometry. Phosphonamide 110 was formed byusing phenoxyphenylamine 14 and allylphosphonic acid chloride methylester instead of allylsulfonyl chloride to result in compound 108(Scheme 22). The rest of synthetic conversion was followed by the samemethod outlined Scheme 21.

When biphenylamine 102 was reacted with allylsulfonyl chloride orallylphosphonic acid chloride methyl ester, compounds III and 115 wereformed, respectively (Scheme 23). Those compounds were converted tocorresponding sulfonamide thiirane 114 and phosphonanide thiirane 118via corresponding diol intermediates 113 and 117.

Biphenylsulfonamide (125, 131) can be made by the reaction betweenaliphatic amines such as compounds 119 or 129 andbiphenylsulfonylchloride (Scheme 24-25). D- or L-Phenylalaninol 119 wasreacted with biphenylsulfonyl chloride 120 in the presence of sodiumbicarbonate (Scheme 24). The resulting alcohol 121 was then mesylatedand then converted to aziridine 122. This aziridine intermediate 122 wasreacted with alkene magnesium bromide to yield compound 123, which wasthen converted to corresponding epoxide 124 and thiirane 125.

Trityl-protected D- or L-phenylalaminol 126 was oxidized under Swernoxidation condition (Scheme 25). The resulting aldehyde 127 was reactedwith triphenylphosphonium methylbromide in the presence of n-BuLi. Thetrityl group in resultant Wittig product 128 was then removed in acidiccondition to afford free amine 129, which was then reacted withbiphenylsulfonyl chloride 120. Conversion of compound 130 to compound131 was followed by the method illustrated in Scheme 24.

Ketones and oximes were considered to be surrogates of the sulfonylgroup (Schemes 26-28). Phenoxyphenylmagnesium bromide was reacted withalkene aldehyde to result in secondary alcohol 133, which was theoxidized to ketone 134 (Scheme 26). The double bond in compound 134 wasconverted to epoxide 135 via hydrobromination. Epoxide 135 thenconverted to thiirane using thiourea. Ketone 136 was transformed tooxime 137 in the presence of hydroxylamine hydrochloride and sodiumacetate.

Phenoxybenzaldehyde 138 was reacted with allylmagnesium chloride to givecompound 139 (Scheme 27). The rest of the functional group conversion tocompound 143 is similar in Scheme 26, except oxidation to ketone wasdone after double bond oxidation and thiirane formation.

Tetrahydro-dibenzofurane moiety was investigated as a surrogate ofphenoxyphenyl group. The synthesis of compound 153 is outlined in Scheme28. Tetrahydrodibenzofuran derivative 149, which was obtained fromphenol and cyclohexene oxide according to literature procedures (J. Am.Chem. Soc. 1935, 57, 2095-2099; Bull. Soc. Chim. Fr. 1956, 1817-1822),was converted 150 under Negishi's conditions (Tetrahedron Lett. 1979,20, 845-848). Epoxidation of 150 was achieved via bromohydrinintermediates and the resulting oxirane 151 was treated with thiourea togive thiirane 152. Finally, the conversion of 152 to oxime 153 wascarried out by the NH₂OH.HCl/AcONa (Tetrahedron Lett. 1989, 30,3471-3472; Chem. Pharm. Bull. 2003, 51, 138-151; Tetrahedron 1968, 24,3347-3360).

Aromatic pyrrol group was also designed as an aromatic scaffold(158-159). Substituted benzaldehyde 154 and ethyl cyanoacetate weresubjected to Knoevenagel condensation in the presence of catalyticamount of piperidine to give α,β-unsaturated cyanoester 155, followed bya tandem Michael addition-decarboxylation involving potassium cyanide.The resulting disuccinonitrile 156 was reduced to form pyrrole 157 usingDibal-H according to the method reported by Babler et al. (TetrahedronLett. 1984, 25, 1659-1660). Pyrrole 157 was deprotonated with sodiumhydride and reacted with several electrophiles which containing oxirane(Synlett 1998, 1411-1413; and Mueller, A. C., Shokal, E. C., (ShellDevelopment Co.), US, 1957). Conversion of oxirane to thiirane was doneunder standard thiourea condition.

Example 3 Experimental Section:

Synthesis—¹H and ¹³C NMR spectra were recorded on either a Varian UnityPlus 300 MHz or a Varian INOVA 500 MHz spectrometer. Chemical shifts arereported in from tetramethylsilane on the 6 scale. Mass spectra wererecorded on a JEOL JMSAX505HA and a Finnigan-MAT 8430 high resolutionmagnetic sector mass spectrometers. For silica gel columnchromatography, EMD Silica gel 60 was employed. Thin-layerchromatography was performed with Whatman 0.25 mm silica gel 60-Fplates. All other reagents were purchased from Aldrich Chemical Company,Lancaster, or Across Organics.

Compounds of Scheme 5.

Compound 2a (R₁═R₂═H). n-BuLi (10.7 mL, 2.5 M in hexane) was added to asolution of 4-bromophenyl phenyl ether (4.7 mL, 26.8 mmol) in anhydrousTHF (120 mL) with vigorous stirring at −78° C. After stirring for 30minutes, sulfur (0.86 g, 26.8 mmol) was added to the reaction mixture.The mixture was stirred for 0.5 hours, while temperature was raised to0° C. After cooled down to −78° C., epichlorohydrin (2.2 mL, 28.1 mmol)was added dropwise to the reaction mixture. Stirring was continued for 1hour, while temperature was raised to −20° C. The reaction was quenchedby the addition of a saturated solution of ammonium chloride. Themixture was extracted with EtOAc and the organic layer was washed withwater, dried (MgSO₄), and filtered. The solvent was evaporated underreduced pressure, and the residue was purified by column chromatographyto afford the desired product (6.70 g, 85%). When allyl bromide oracetyl chloride were used instead of epichlorohydrin for this reaction,compound 10a or 11a were obtained; ¹H NMR (500 MHz, CDCl₃) δ 7.4 (d,J=8.4 Hz, 2H), 7.4 (dd, J=8.4, 7.6 Hz, 2H), 7.1 (t, J=7.4 Hz, 1H), 7.0(d, J=7.6 Hz, 2H), 7.0 (d, J=8.6 Hz, 2H), 3.9 (m, 1H, CHOH), 3.7 (m, 2H,CH₂Cl), 3.1 (ddd, J=42.5, 14.0 Hz, 5.6 Hz, 2H, SCH₂CH), 2.8 (brs, 1H,OH); ¹³C NMR (125 MHz, CDCl₃) δ 157.2, 156.7, 133.2, 130.0, 128.0,123.9, 119.4, 119.3, 69.6, 48.1, 39.7; HRMS calcd for C₁₅H₁₅ClO₂S (M⁺)294.0481, found 294.0465.

Compound 3a (R₁═R₂═H). Potassium carbonate (2.81 g, 20.4 mmol) was addedto a solution of compound 2a (3.00 g, 10.2 mmol) in a 1:2 mixture ofmethanol and acetonitrile (100 mL) in ice-water bath with vigorousstirring. After 10 minutes, ice-water bath was removed and stirring wascontinued for 1 hour at room temperature and was filtered through asmall layer of silica gel. The filtrate was concentrated under reducedpressure, and the residue was purified by column chromatography toafford the desired product (2.49 g, 95%) as an oil; ¹H NMR (500 MHz,CDCl₃) δ 7.4 (d, J=8.9 Hz, 2H), 7.4 (t, J=8.0 Hz, 2H), 7.1 (t, J=7.4 Hz,1H), 7.0 (d, J=8.6 Hz, 2H), 7.0 (d, J=8.8 Hz, 2H), 3.2 (m, 1H), 3.1 (dd,J=14.2, 5.2 Hz, 1H), 2.9 (dd, J=14.0, 6.0 Hz, 1H), 2.8 (t, J=4.8 Hz,1H), 2.5 (dd, J=5.0, 2.6 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 157.0,156.7, 133.5, 129.9, 128.7, 123.8, 119.3, 119.2, 51.2, 47.4, 38.0; HRMScalcd for C₁₅H₁₄O₂S (M⁺) 258.0715, found 258.0729.

Compound 4a (R₁═R₂═H). To a solution of compound 3a (2.00 g, 7.74 mmol)in dichloromethane (20 mL) was added a solution of m-chloroperoxybenzoicacid (3.47 g, 15.5 mmol, 77%) in ice-water bath. After 10 minutes, thesuspension was filtered, and the filtrate was diluted with EtOAc andwashed with 10% aqueous sodium thiosulfate, followed by saturated sodiumbicarbonate and brine. The organic layer was dried over magnesiumsulfate and was concentrated. The product was purified by silica gelchromatography to yield the title compound as an oil (1.96 g, 87%); ¹HNMR (500 MHz, CDCl₃) δ 7.9 (d, J=8.8 Hz, 2H), 7.4 (dd, J=8.4, 7.4 Hz,2H), 7.2 (t, J=6.9 Hz, 1H), 7.1 (d, J=8.6 Hz, 4H), 3.3 (m, 3H), 2.8 (m,1H), 2.5 (dd, J=5.2, 4.2 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 163.0,154.8, 132.5, 130.6, 130.4, 125.4, 120.6, 117.7, 59.7, 46.0, 46.0; HRMScalcd for C₁₅H₁₄O₄S (M⁺) 290.0613, found 290.0630.

Compound 5a (R₁═R₂═H). Thiourea (0.52 g, 6.89 mmol) was added to asolution of compound 4a (1.00 g, 3.44 mmol) in methanol (10 mL). Thereaction mixture was stirred at room temperature overnight. The solventwas removed under reduced pressure. The residue was partitioned betweenethyl ether and water, the organic layer was washed with water andbrine, dried (MgSO₄), and the suspension was filtered. Evaporation ofsolvent gave the crude product, which was purified by columnchromatography. The desired product was crystallized as white needlesfrom 1-butanol (0.89 g, 84%); ¹H NMR (500 MHz, CDCl₃) δ 7.9 (d, J=8.6Hz, 2H), 7.5 (t, J=7.8 Hz, 2H), 7.3 (t, J=7.2 Hz, 1H), 7.1 (d, J=8.6 Hz,4H), 3.6 (dd, J=14.2, 5.6 Hz, 1H), 3.2 (dd, J=14.2, 7.8 Hz, 1H), 3.1 (m,1H), 2.6 (dd, J=6.0, 1.4 Hz, 1H), 2.2 (dd, J=5.2, 1.6 Hz, 1H); ¹³C NMR(125 MHz, CDCl₃) δ 163.1, 154.9, 132.0, 130.9, 125.5, 120.6, 117.9,62.8, 26.3, 24.4; HRMS calcd for C₁₅H₁₄O₃S₂ (M⁺) 306.0384, found306.0396.

Compounds of Scheme 6.

Compound 4a (from compound 10a). Compound 10a (2.40 g, 10.0 mmol) wasdissolved in m-CPBA (11.5 g, 50.0 mmol, 77% max) in ice-water bath andstirred for 7 days. The resulting suspension was filtered and filtratewas diluted with EtOAc and washed with 10% aqueous sodium thiosulfate,followed by saturated sodium bicarbonate and brine. The organic layerwas dried over magnesium sulfate and was concentrated. The product waspurified by silica gel chromatography to yield the title compound as anoil (2.50 g, 87%).

Compound 12a (R₁═R₂═H). A mixture of compound 10a (0.27 g, 1.1 mmol) andN-methylmorpholine N-oxide (0.25 g, 2.1 mmol) in acetone-water (30 mL,4:1) was treated with osmium tetraoxide (0.3 mL, 47 μmol, 4% aqueoussolution) and the resultant solution was stirred at room temperatureovernight under dark. Sodium sulfite (0.1 g) was added and the resultingmixture was stirred for an additional 1 hour. After filtration through asmall layer of silica gel, the filtrate was evaporated and the residuewas purified by column chromatography on silica gel to give the titlecompound (0.26 g, 85%); ¹H NMR (300 MHz, CDCl₃): δ 7.4 (d, J=8.4 Hz,2H), 7.4 (dd, J=8.4, 7.6 Hz, 2H), 7.1 (t, J=7.4 Hz, 1H), 7.0 (d, J=7.6Hz, 2H), 7.0 (d, J=8.6 Hz, 2H), 3.83 (brs, 1H), 3.51-3.68 (m, 2H),3.15-3.25 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 157.2, 156.7, 133.2,130.0, 128.0, 123.9, 119.4, 119.3, 69.6, 48.1, 39.7.

Compound 13a (R₁═R₂═H). Compound 12a was oxidized to compound 13a by thesame procedure for the preparation of compound 4a from compound 3a.

Compound 4a (from compound 13a). Conversion of diol to oxirane was doneby two methods. The first method is using Mitsunobu condition. Thesecond is via the formation of tosylated intermediate. A mixture ofcompound 13a (163 mg, 530 μmol), triphenylphosphine (142 mg, 530 μmol),and diethylazodicarboxylate (90 μL, 530 μmol) was refluxed for 4 hoursin anhydrous benzene (10 mL) and the volatile was evaporated. Theresidue was purified by column chromatography on silica gel to affordthe title compound (98.5 mg, 64%).

The second procedure involving formation of a tosylated intermediate: Amixture of compound 13a (163 mg, 530 μmol) and tosyl chloride (111 mg,583 μmol) in CH₂Cl₂ (2 mL) was treated with pyridine (138 μL, 1.75 mmol)and the resulting mixture was stirred overnight in ice-water bath. Thereaction mixture was washed with water several times and volatile wasconcentrated. The crude product was used for the next step withoutfurther purification was dissolved in anhydrous THF (2 mL) and cooleddown in ice-water bath. To this reaction mixture, NaH (35 mg, 875 μmol,60% in oil) was added and the resulting mixture was stirred for 10minutes and filtered through a small layer of silica gel and washed withTHF. The combined filtrate was evaporated under reduced pressure and theresulting crude product was purified by column chromatography to affordthe desired product as colorless oil (115 mg, 75%).

Compound 3a (from compound 11a). Compound 11a (0.61 g, 2.5 mmol) wasstirred at room temperature for 0.5 hours in the presence of K₂CO₃ (1.38g, 20 mmol) in 1:1 mixture of MeOH:acetonitrile (10 mL). Epibromohydrin(0.8 mL, 10.0 mmol) was added dropwise to the reaction mixture and theresulting solution was stirred at room temperature for additional 1hour. The reaction mixture was filtered and the filtrate was evaporated.The residue was purified by column chromatography on silica gel toafford the desired product (0.56 g, 87%). When allyl bromide or glycidolwere used for this reaction instead of epichlorohydrin, compound 10a or12a were obtained in similar yield.

Compounds of Scheme 7.

Compound 11b (from compound 14a, R₁═R₂═H, 3-amino). A solution ofo-benzenedisulfonimide (2.63 g, 12 mmol) in glacial acetic acid (40 mL)was slowly added, over a period of 10 minutes, to a solution of3-phenoxyaniline (1.85 g, 10 mmol) in acetic acid (20 mL) in an icebath. Isoamylnitrite (1.5 mL, 11 mmol) was added dropwise to thereaction mixture over 10 min and the resulting mixture was stirred for0.5 hours at the same temperature. Addition of diethyl ether to thereaction mixture resulted in diazonium salt as an orange powder, whichwas filtered and washed with cold diethyl ether. Potassium thioacetate(0.98 g, 10 mmol) in acetonitrile (10 mL) was added to the crudediazonium salt (2.0 g) in anhydrous acetonitrile (10 mL) and theresulting suspension was stirred at room temperature for 2 hours. Thereaction mixture was filtered and the filtrate was evaporated. Theresidue was purified by column chromatography on silica gel to affordthe desired product (0.97 g, 40%); ¹H NMR (500 MHz, CDCl₃): δ 2.45 (s,3H), 7.09-7.22 (m, 6H), 7.38-7.44 (m, 3H); ¹³C NMR (125 MHz, CDCl₃): δ30.3, 119.4, 119.8, 123.9, 124.4, 129.1, 130.4, 156.7, 157.9; HRMS (FAB)calcd for C₁₄H₂O₂S (M⁺) 244.0558, found 244.0556.

Compound 3b (from compound 11b, R₁═R₂═H, 2-amino). Compound 11b (0.61 g,2.5 mmol) was stirred at room temperature for 0.5 hours in the presenceof K₂CO₃ (1.38 g, 10 mmol) in 1:1 mixture of MeOH: acetonitrile (10 mL).Epibromohydrin (0.8 mL, 10.0 mmol) was added dropwise to the reactionmixture and the resulting solution was stirred at room temperature foradditional 1 hour. The reaction mixture was filtered and the filtratewas evaporated. The residue was purified by column chromatography onsilica gel to afford the desired product (0.56 g, 87%); ¹H NMR (500 MHz,CDCl₃): δ 2.57 (dd, 1H, J=2.2, 4.7 Hz), 2.81 (t, 1H, J=4.2 Hz), 2.98 (q,1H, J=7.8 Hz), 3.16-3.20 (m, 2H), 6.88 (dd, 1H, J=2.5, 7.9 Hz),7.04-7.09 (m, 3H), 7.16 (t, 2H, J=7.4 Hz), 7.28 (t, 1H, J=−7.8 Hz), 7.38(t, 2H, J=7.8 Hz); ¹³C NMR (125 MHz, CDCl₃): δ 36.3, 47.6, 51.0, 117.0,119.3, 119.8, 123.8, 124.4, 130.0, 130.3, 137.4, 156.8, 157.9; HRMS(FAB) calcd for C₁₅H₁₄O₂S (M⁺) 258.0715, found 258.0713.

Conversion from compound 3b to compounds 4b, and 5b was followed by thesame method as described in Scheme 5.

Compound 4b (R₁═R₂═H, 2-amino). ¹H NMR (500 MHz, CDCl₃): δ 2.49 (dd, 1H,J=1.0, 5.0 Hz), 2.84 (dd, 1H, J=3.7, 4.7 Hz), 3.26-3.37 (m, 3H), 7.08(dd, 1H, J=1.2, 8.7 Hz), 7.22 (t, 1H, J=7.4 Hz), 7.32 (m, 1H), 7.42 (dd,2H, J=7.4, 8.4 Hz), 7.56 (m, 2H), 7.68 (dt, 1H, J=1.0, 8.4 Hz); ¹³C NMR(125 MHz, CDCl₃): δ 45.9, 46.1, 59.6, 117.7, 119.8, 122.5, 124.0, 124.9,130.4, 131.0, 140.8, 155.9, 158.6; HRMS (FAB) calcd for C₁₅H₁₄O₄S (M⁺)290.0613, found 290.0603.

Compound 5b (R₁═R₂═H, 2-amino). ¹H NMR (500 MHz, CDCl₃): δ 2.14 (dd, 1H,J=1.7, 5.2 Hz), 2.51 (dd, 1H, J=1.7, 6.2 Hz), 3.03 (m, 1H), 3.18 (dd,1H, J=7.7, 14.1 Hz), 3.51 (dd, 1H, J=5.4, 14.3 Hz), 7.05 (dd, 2H, J=1.0,8.9 Hz), 7.19 (t, 1H, J=7.4 Hz), 7.29, 7.31 (2dd, 1H, J=1.0, 2.5 Hz),7.39 (dd, 2H, J=7.4, 8.4 Hz), 7.52-7.55 (m, 2H), 7.64 (dt, 1H, J=1.5,7.9 Hz); ¹³C NMR (125 MHz, CDCl₃): δ 24.3, 20.0, 62.4, 117.7, 119.8,122.6, 123.9, 124.8, 130.3, 131.0, 140.2, 155.7, 158.6; HRMS (FAB) calcdfor C₁₅H₁₅O₃S₂ (MH⁺) 307.0463, found 307.0474.

Compounds of Scheme 8.

Compound 1b (R₁═H, R₂=d4, from compound 16a and 17a). The procedure wasadapted from that reported by Ma et al. (Org. Lett. 2003, 5, 3799-3802).A mixture of 1,4-dibromobenzene-d₄ (17a, 2.50 g, 10.4 mmol), phenol(16a, 1.47 g, 15.6 mmol), Cs₂CO₃ (6.80 g, 20.9 mmol),N,N-dimethylglycine hydrochloride salt (0.44 g, 3.15 mmol), CuI (0.20mg, 1.05 mmol) in degassed 1,4-dioxane (20 mL) was heated at 90° C. for22 hours under a nitrogen atmosphere. After dilution with water, themixture was extracted with ethyl acetate. The combined organic layer waswashed with water and brine, dried over anhydrous MgSO₄, andconcentrated under reduced pressure. The resultant residue was purifiedby column chromatography to give the desired product as colorless oil(2.00 g, 76%). ¹H NMR (500 MHz, CDCl₃) δ 7.06 (d, J=7.9 Hz, 2H), 7.18(t, J=7.6 Hz, 1H), 7.39 (t, J=7.2 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ115.5, 119.2, 120.2 (t, J=25.5 Hz), 123.9, 130.1, 132.4 (t, J=25.5 Hz),156.7, 156.9; HRMS calcd for C₁₂H₅D₄BrO (M⁺) 252.0088, found 252.0072.

Conversion from compound 1b to compounds 2c-5c was followed by the samemethod as described in Scheme 5.

Compound 2c (R₁═H, R₂=d4) ¹H NMR (500 MHz, CDCl₃) δ 3.03 (dd, J=13.8,7.2 Hz, 1H), 3.12 (dd, J=14.1, 5.5 Hz, 1H), 3.64-3.74 (m, 2H), 3.88-3.94(m, 1H), 7.03 (d, J=7.6 Hz, 2H), 7.15 (t, J=7.2 Hz, 1H), 7.36 (t, J=8.6,7.6 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 39.8, 48.2, 69.6, 119.1 (t,J=25.5 Hz), 119.4, 124.0, 127.7, 130.1, 132.9 (t, J=25.5, 23.9 Hz),156.7, 157.3; HRMS calcd for C₁₅H₁₁D₄ClO₂S (M⁺) 298.0732, found298.0737.

Compound 3c (R₁═H, R₂=d4). ¹H NMR (500 MHz, CDCl₃) δ 2.49 (dd, J=4.1,1.4 Hz, 1H), 2.82-2.86 (m, 1H), 3.28-3.36 (m, 3H), 7.11 (dd, J=8.6, 0.7Hz, 2H), 7.27 (t, J=7.6 Hz, 1H), 7.45 (t, J=8.3, 7.6 Hz, 2H); ¹³C NMR(126 MHz, CDCl₃) δ 38.2, 47.6, 51.3, 119.0 (t, J=25.7 Hz), 119.3, 123.9,128.5, 130.0, 133.2 (t, J=23.9 Hz), 156.8, 157.1 (m); HRMS calcd forC₁₅H₁₀D₄O₂S (M⁺) 262.0966, found 262.0949.

Compound 4c (R₁═H, R₂=d4). ¹H NMR (500 MHz, CDCl₃) δ 2.50 (dd, J=4.1,1.4 Hz, 1H), 2.84 (dd, J=3.8, 2.4 Hz, 1H), 3.26-3.37 (m, 3H), 7.11 (d,J=8.6 Hz, 2H), 7.27 (t, J=7.4 Hz, 1H), 7.45 (t, J=7.9 Hz, 2H); ¹³C NMR(126 MHz, CDCl₃) δ 46.0, 46.1, 59.8, 117.4 (t, J=25.5 Hz), 120.7, 125.4,130.3 (t, J=25.5 Hz), 130.4, 132.4, 154.9, 163.0; HRMS calcd forC₁₅H₁₀D₄O₄S (M⁺) 294.0864, found 294.0869.

Compound 5c (R₁═H, R₂=d4). ¹H NMR (500 MHz, CDCl₃) δ 2.17 (dd, J=5.2,1.7 Hz, 13H), 2.54 (dd, J=6.2, 1.7 Hz, 1H), 3.04-3.10 (m, 1H), 3.18 (dd,J=14.5, 7.9 Hz, 1H), 3.53 (dd, J=14.1, 5.9 Hz, 1H), 7.10 (d, J=7.6 Hz,2H), 7.25 (t, J=6.9 Hz, 1H), 7.44 (t, J=7.9 Hz, 2H); ¹³C NMR (126 MHz,CDCl₃) δ24.4, 26.3, 62.8, 117.5 (t, J=24.7 Hz), 120.6, 125.4, 125.4,130.5 (t, J=25.5 Hz), 130.5, 131.9, 154.9, 163.0; HRMS calcd forC₁₅H₁₁D₄O₃S₂ M+H⁺) 311.0714, found 311.0700.

Compound 19a (R₂═H). To a stirred solution of 4-hydroxythiophenol (6)(4.30 g, 34.1 mmol) in DMF (25 mL) were added K₂CO₃ (4.71 g, 34.1 mmol)and allyl bromide (3.09 mL, 34.1 mmol) at ice-water temperature, and themixture was stirred for 15 minutes, prior to stirring overnight at roomtemperature. After the addition of 1 M aqueous HCl, the mixture wasextracted with ether (3×). The combined organic layer was washed withwater and brine, dried over MgSO₄, and concentrated under reducedpressure. The resultant residue was purified by silica gel columnchromatography (ethyl acetate/hexane= 1/10 to ⅙) to give the product(5.74 g, 70%) as a white semi-solid.

Compound 10b (R₁=p-CH₂CO₂Et, R₂═H). A mixture of 18a (1.51 g, 6.59mmol), 19a (1.64 g, 9.88 mmol), Cs₂CO₃ (4.30 g, 13.2 mmol),N,N-dimethylglycine hydrochloride salt (276 mg, 1.98 mmol), CuI (125 mg,0.659 mmol), and degassed 1,4-dioxane (14 mL) was heated at 90° C. for22 hours under a nitrogen atmosphere. After dilution with water, themixture was extracted with ethyl acetate. The combined organic layer waswashed with water and brine, dried over Na₂SO₄, and concentrated underreduced pressure. The resultant residue was purified by silica gelcolumn chromatography (ethyl acetate/hexane=1/12) to give the product(1.35 g, 65%) as a pale yellow semi-solid. ¹H NMR (300 MHz, CDCl₃): δ3.49 (d, 2H, J=7.2 Hz), 3.61 (s, 2H), 3.71 (s, 3H), 5.03-5.10 (m, 2H),5.86 (m, 1H), 6.91-6.97 (m, 4H), 7.23-7.26 (m, 2H), 7.32-7.35 (m, 2H);¹³C NMR (125 MHz, CDCl₃): δ38.5, 40.3, 52.1, 117.5, 119.0, 119.1, 129.0,129.3, 130.6, 132.9, 133.7, 156.0, 156.3, 172.0; HRMS (FAB) calcd forC₁₈H₁₈O₃S (M⁺) 314.0977, found 314.0986.

Compound 20a (R₂═H). Synthesis of compound 20a was accomplished by thesame method for the preparation of compound 19a using dimethylcarbamoylchloride instead of allyl bromide.

Compound 21a (R₁=3-benzyloxy, R₂=1). Synthesis of compound 21a wasaccomplished by the same method for the preparation of compound 10busing compound 20a and 1-benzyloxy-3-iodobenzene (18b). ¹H NMR (500 MHz,CDCl₃) δ 3.09 (d, J=23.3 Hz, 6H), 5.06-5.08 (m, 2H), 6.70 (dd, J=8.2,2.4 Hz, 1H), 6.75 (t, J=2.2 Hz, 1H), 6.82 (dd, J=8.4, 2.4 Hz, 1H), 7.05(d, J=9.0 Hz, 2H), 7.28 (t, J=8.2 Hz, 1H), 7.34-7.51 (m, 7H); ¹³C NMR(126 MHz, CDCl₃) δ 36.9, 70.1, 106.4, 110.6, 111.9, 118.9, 127.6, 128.1,128.6, 130.3, 136.7, 137.5, 157.5, 158.4, 160.1, 167.2.

Compound 3d (R₁=3-benzyloxy, R₂═H, from compound 21a). Compound 21a(4.30 g, 11.3 mmol) was added to methanolic KOH (6.26 g, 88.2 mol) inMeOH (80 mL) and then refluxed for 3 hours. The solvent was removedunder reduced pressure and the residue was diluted with methylenechloride/2 N HCl. The aqueous layer was extracted with methylenechloride and the combined organic layer was washed with water and brine,dried over anhydrous MgSO₄, and concentrated under reduced pressure. Theresultant residue was dissolved in acetonitrile/MeOH (2:1, 60 mL) andK₂CO₃ (2.95 g, 21.4 mmol) was added. After 0.5 hours, epichlorohydrin(1.67 mL, 21.4 mmol) was added to the reaction mixture and was stirredat room temperature for 1 hour and was filtered through a layer ofsilica gel. The filtrate was evaporated under reduced pressure and theresidue was purified by column chromatography to give compound 3d ascolorless oil (3.00 g, 73%). ¹H NMR (500 MHz, CDCl₃) δ 3.09 (d, J=24.1Hz, 6H), 5.04-5.08 (m, 2H), 6.69 (dd, J=7.2, 3.1 Hz, 1H), 6.74 (t, J=2.4Hz, 1H), 6.81 (dd, J=8.3, 2.4 Hz, 1H), 7.04 (d, J=9.0 Hz, 2H), 7.28 (t,J=8.3 Hz, 1H), 7.34-7.50 (m, 7H); ¹³C NMR (126 MHz, CDCl₃) δ 70.2,106.4, 110.6, 112.0, 118.9, 122.4, 127.6, 128.1, 128.7, 130.4, 136.7,137.5, 157.5, 158.4, 160.2, 167.2.

Compound 23a (R₂═H). Synthesis of compound 23a was accomplished by thesame method for the preparation of compound 19a using epichlorohydrininstead of allyl bromide. ¹H NMR (500 MHz, CDCl₃) δ 2.95 (dd, J=13.8,7.2 Hz, 1H), 3.03 (dd, J=13.8, 5.5 Hz, 1H), 3.25 (br.s., 1H), 3.66 (ddd,J=21.4, 11.0, 4.1 Hz, 2H), 3.87 (m, 1H), 6.79 (d, J=8.3 Hz, 2H), 7.22(br.s., 1H), 7.31 (d, J=8.3 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 40.3,48.0, 69.6, 116.4, 124.0, 134.3, 156.4, 172.2; HRS calcd for C₉H₁₁ClO₂S(M⁺) 218.0168, found 218.0172.

Compound 2e (R₂=d5, R₂═H, from compound 22a and compound 23a). Theprocedure was adapted from that reported by Evan et al. (TetrahedronLett. 1998, 39, 2937-2940). A mixture of compound 23a (0.86 g, 3.93mmol), Cu(OAc)₂ (0.72 g, 3.96 mmol), phenyl-d₅ boronic acid (22a, 1.00g, 7.87 mmol), and powdered 4 Å molecular sieves was stirred in CH₂Cl₂and triethylamine (1.10 mL, 7.89 mmol) was added. After stirring for 18hours at room temperature, the resulting slurry was filtered through alayer of Celite and the filtrate was concentrated under reducedpressure. The residue was purified by column chromatography to give thedesired product (0.85 g, 72%). ¹H NMR (500 MHz, CDCl₃) δ 2.95 (br.s.,1H), 3.09 (ddd, J=42.0, 14.1, 5.5 Hz, 2H), 3.71 (ddd, J=17.2, 11.0, 4.5Hz, 2H), 3.94 (m, 1H), 6.98 (d, J=9.0 Hz, 2H), 7.43 (d, J=9.0 Hz, 2H);¹³C NMR (126 MHz, CDCl₃) δ 39.6, 48.1, 69.6, 118.9 (t, J=24.7 Hz),119.4, 123.4 (t, J=24.7 Hz), 128.0, 129.5 (t, J=23.9 Hz), 133.2, 156.6,157.2; HRMS calcd for C₁₅H₁₀D₅ClO₂S (M⁺) 299.0795, found 299.0988.

Conversion from compound 2e to compounds 3e-5e was followed by the samemethod as described in Scheme 5.

Compound 3e (R₂=d5, R₂═H). ¹H NMR (500 MHz, CDCl₃) δ 2.50 (dd, J=4.8,2.8 Hz, 1H), 2.79 (td, J=8.6, 0.7 Hz, 1H), 2.89 (dd, J=13.8, 5.9 Hz,1H), 3.12 (dd, J=13.8, 5.2 Hz, 1H), 3.16-3.21 (m, 1H), 6.96 (d, J=9.0Hz, 22H), 7.45 (d, J=9.0 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 38.1, 47.6,51.3, 118.9 (t, J=25.5 Hz), 119.3, 123.3 (t, J=23.9 Hz), 128.7, 129.5(t, J=24.0 Hz), 133.6, 156.7, 157.2; HRMS calcd for C₁₅H₉D₅O₂S (M⁺)263.1028, found 263.1035.

Compound 4e (R₂=d5, R₂═H). ¹H NMR (500 MHz, CDCl₃) δ 2.48 (dd, J=4.8,2.1 Hz, 1H), 2.81-2.84 (m, 1H), 3.24-3.36 (m, 3H), 7.09 (d, J=9.0 Hz,2H), 7.89 (d, J=9.0 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 45.7, 45.8,59.5, 117.6, 120.0 (t, J=24.7 Hz), 124.7 (t, J=23.9 Hz), 129.7 (t,J=24.7 Hz), 130.5, 132.4, 154.6, 162.7; HRMS calcd for C₁₅H₉D₅O₄S (M⁺)295.0926, found 295.0929.

Compound 5e (R₂-d₅, R₂═H). ¹H NMR (500 MHz, CDCl₃) δ 2.16 (dd, J=5.2,1.7 Hz, 1H), 2.54 (dd, J=6.2, 1.4 Hz, 1H), 3.03-3.10 (m, 1H), 3.18 (dd,J=14.1, 7.9 Hz, 1H), 3.53 (dd, J=14.1, 5.5 Hz, 1H), 7.10 (d, J=9.0 Hz,2H), 7.87 (d, J=9.0 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 24.4, 26.3,62.8, 117.9, 120.2 (t, J=23.9 Hz), 124.9 (t, J=25.0 Hz), 129.9 (t,J=24.7 Hz), 130.9, 132.0, 154.8, 163.1; HRMS calcd for C₁₅H₁₀D₅O₃S₂(M+H⁺) 312.0773, found 312.0796.

Compounds of Scheme 9.

Compound 28a (R₂=p-NO₂, Rz=allylthio). To a stirred solution of 19a(3.46 g, 20.8 mmol) in DMF (100 ml) were added cesium carbonate (10.2 g,31.2 mmol) and 1-fluoro-4-nitrobenzene (25a) (2.94 g, 20.8 mmol) at roomtemperature, and the mixture was stirred at the same temperature for 2days. After dilution with water, the mixture was extracted into hexane(3×). The combined organic layer was washed with water and brine, driedover Na₂SO₄, and concentrated under reduced pressure to give 28a (5.32g, 89%) as a pale yellow oil. ¹H NMR (300 MHz, CDCl₃): δ 3.55 (dt, 2H,J=6.9, 1.2 Hz), 5.10 (dt, 1H, J=10.2, 1.2 Hz), 5.13 (dt, 1H, J=17.1, 1.2Hz), 5.88 (ddt, 1H, J=17.1, 10.2, 6.9 Hz), 6.98-7.04 (m, 4H), 7.38-7.43(m, 2H), 8.18-8.22 (m, 2H); ¹³C NMR (125 MHz, CDCl₃): δ 37.8, 117.1,117.9, 120.9, 126.0, 132.3, 132.5, 133.4, 142.7, 153.4, 163.1; HRMS(FAB) calcd for C₁₆H₁₇NO₃S₂ (M⁺) 287.0616, found 287.0593.

Compound 29a (R₂=p-NO₂, R₂=dimethylthiocarbamyl). Preparation ofcompound 29 was accomplished by the method for the synthesis of compound28a using 2-chloro-5-nitropyridine (26a) and compound 20a. ¹H NMR (500MHz, CDCl₃) δ 3.12 (d, J=33.3 Hz, 6H), 7.06 (d, J=9.0 Hz, 1H), 7.19 (d,J=8.6 Hz, 2H), 7.58 (d, J=8.6 Hz, 2H), 8.49 (m, 1H), 9.04 (d, J=1.4 Hz,6H); ¹³C NMR (126 MHz, CDCl₃) δ 37.2, 111.7, 122.2, 125.7, 126.4, 135.2,137.5, 145.1, 153.7, 166.7 (s); HRMS calcd for C₂₅H₃₀N₅O₆S (M+H⁺)528.1917, found 528.1920.

Compounds of Scheme 10.

Compound 34a (R₁═R₂═H, R₃=Me, n=1,4-carbonyloxy). A mixture of compound33a (100 mg, 310 μmol) and pyridine (50 μL, 620 μmol) in CH₂Cl₂ (1 mL)in ice-water bath was added acetic anhydride (32 μL, 340 μmol) and theresulting solution was stirred for 1 hour. The reaction mixture washedwith water and the volatile was concentrated and the crude product waspurified by column chromatography. ¹H NMR (500 MHz, CDCl₃) δ 2.15 (dd,J=4.8, 1.4 Hz, 1H), 2.31 (s, 3H), 2.52 (d, J=6.2 Hz, 1H), 3.05 (m, 1H),3.18 (dd, J=14.3, 7.8 Hz, 1H), 3.51 (dd, J=14.3, 5.7 Hz, 1H), 7.07-7.16(m, 6H), 7.86 (d, J=9.0 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ21.2, 24.3,26.2, 62.7, 117.8, 121.4, 123.5, 130.9, 132.2, 147.7, 152.3, 162.8,169.5.

Compounds 34b-34h were prepared in the same manner as described for 34a,with the exception corresponding acid chlorides or anhydrides was usedin place of acetic anhydride.

Compound 34b (R₁═R₂═H, R₃=t-Bu, n=1,4-carbonyloxy). ¹H NMR (500 MHz,CDCl₃) δ 1.40 (s, 9H), 2.17 (dd, J=5.2, 1.7 Hz, 1H), 2.55 (dd, J=6.2,1.7 Hz, 1H), 3.08 (m, 1H), 3.19 (dd, J=14.3, 7.8 Hz, 1H), 3.53 (dd,J=14.1, 5.5 Hz, 1H), 7.09-7.14 (m, 6H), 7.87 (d, J=9.0 Hz, 2H); ¹³C NMR(126 MHz, CDCl₃) δ 24.4, 26.3, 27.3, 39.3, 62.8, 117.8, 121.5, 123.5,130.9, 132.2, 148.2, 152.1, 163.1, 177.3.

Compound 34c (R₁═R₂═H, R₃=Ph, n=1,4-carbonyloxy). ¹H NMR (500 MHz,CDCl₃) δ 2.17 (dd, J=5.2, 1.7 Hz, 1H), 2.54 (dd, J=4.5, 1.7 Hz, 1H),3.04-3.09 (m, 1H), 3.20 (dd, J=14.3, 7.8 Hz, 1H), 3.53 (dd, J=14.3, 5.7Hz, 1H), 7.15 (t, J=8.6 Hz, 4H), 7.29 (d, J=9.0 Hz, 2H), 7.53 (t, J=7.8Hz, 2H), 7.66 (t, J=7.6 Hz, 1H), 7.89 (d, J=9.0 Hz, 2H), 8.22 (d, J=7.2Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 24.4, 26.2, 62.7, 117.8, 121.5,123.7, 128.8, 129.3, 130.3, 130.9, 132.3, 134.0, 148.0, 152.4, 162.9,165.3.

Compound 34d (R₁═R₂═H, R₃═CH₂Ph, n−1,4-carbonyloxy). ¹H NMR (500 MHz,CDCl₃) δ 2.17 (m, 1H), 2.48 (s, 2H), 2.55 (t, J=6.0 Hz, 1H), 3.08 (m,1H), 3.19 (m, 1H), 3.52 (m, 1H), 3.89 (s, 1H), 7.09 (t, J=8.6 Hz, 2H),7.12-7.17 (m, 3H), 7.26-7.35 (m, 3H), 7.37-7.42 (m, 2H), 7.88 (ddd,J=11.8, 8.9, 2.8 Hz, 2H), 8.11 (d, J=7.9 Hz, 1H).

Compound 34e (R₁═R₂═H, R₃═CH₁₂(CH₂)₉Br, n=1,4-carbonyloxy). ¹H NMR (500MHz, CDCl₃) δ 1.57 (m, 2H), 1.78 (m, 2H), 1.92 (m, 2H), 2.14 (dd, J=5.2,1.7 Hz, 1H), 2.52 (d, J=6.2 Hz, 1H), 2.59 (t, J=7.4 Hz, 3H), 3.04 (m,1H), 3.18 (dd, J=14.1, 7.9 Hz, 1H), 3.43 (t, J=6.5 Hz, 3H), 3.50 (dd,J=14.3, 5.7 Hz, 1H), 7.07-7.11 (m, 4H), 7.12-7.15 (m, 2H), 7.86 (d,J=9.0 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 24.1, 24.3, 26.2, 27.6, 32.4,33.6, 34.1, 62.7, 117.8, 121.4, 123.4, 130.9, 132.2, 147.7, 152.2,162.9, 172.0.

Compound 34f (R₁═R₂═H, R₃=4-methylphenyl, n=1,4-carbonyloxy). ¹H NMR(500 MHz, CDCl₃) δ 2.18 (d, J=4.1 Hz, 1H), 2.47 (s, 3H), 2.56 (d, J=5.9Hz, 1H), 3.08 (m, 1H), 3.19 (dd, J=14.1, 7.6 Hz, 1H), 3.54 (dd, J=14.1,5.5 Hz, 1H), 7.15 (dd, J=8.8, 5.0 Hz, 4H), 7.30 (dd, J=23.1, 7.9 Hz,4H), 7.89 (d, J=9.0 Hz, 2H), 8.11 (d, J=8.3 Hz, 2H); ¹³C NMR (126 MHz,CDCl₃) δ 22.0, 24.4, 26.3, 62.8, 117.9, 121.6, 123.8, 126.6, 129.6,130.4, 131.0, 132.3, 144.9, 148.1, 152.3, 163.1, 165.4.

Compound 34g (R₁═R₂₁═H, R₃=4-nitrophenyl, n=1,4-carbonyloxy). ¹H NMR(500 MHz, CDCl₃) δ2.18 (dd, J=5.0, 1.9 Hz, 1H), 2.56 (d, J=6.2 Hz, 1H),3.08 (m, 1H), 3.22 (dd, J=14.3, 7.8 Hz, 1H), 3.53 (dd, J=14.5, 5.9 Hz,1H), 7.17 (dd, J=14.8, 9.0 Hz, 4H), 7.32 (d, J=9.0 Hz, 1H), 7.91 (d,J=9.0 Hz, 2H), 8.34-8.43 (m, 4H); ¹³C NMR (126 MHz, CDCl₃) δ24.4, 26.3,62.8, 118.1, 121.7, 123.5, 124.0, 124.4, 131.1, 131.5, 131.9, 132.6,134.8, 147.5, 151.2, 152.9, 162.8, 163.5.

Compound 34h (R₁═R₂═H, R₃=4-methoxyphenyl, n=1,4-carbonyloxy). ¹H NMR(500 MHz, CDCl₃) δ 2.18 (dd, J=5.2, 1.7 Hz, 1H), 2.56 (dd, J=6.2, 1.0Hz, 1H), 3.08 (m, 1H), 3.19 (dd, J=14.3, 7.8 Hz, 1H), 3.54 (dd, J=14.3,5.7 Hz, 1H), 3.92 (s, 3H), 7.01 (d, J=9.0 Hz, 2H), 7.15 (dd, J=9.0, 3.8Hz, 4H), 7.28 (d, J=9.0 Hz, 2H), 7.89 (d, J=9.0 Hz, 2H), 8.17 (d, J=9.0Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 24.4, 26.3, 55.8, 62.8, 114.1,117.9, 121.6, 121.6, 123.8, 131.0, 132.3, 132.5, 148.2, 152.3, 163.1,164.3, 165.1.

Compound 35a (R₁═R₂═H, R₃=Me, n=1,4-sulfonyloxy). A mixture of compound33a (100 mg, 310 μmol) and Et₃N (86 μL, 620 mmol) in CH₂Cl₂ (1 mL) inice-water bath was added acetic anhydride (26 μL, 340 μmol) and theresulting solution was stirred for 1 hour. The reaction mixture washedwith water and the volatile was concentrated and the crude product waspurified by column chromatography. ¹H NMR (500 MHz, CDCl₃) δ 2.13 (dd,J=5.2, 1.7 Hz, 1H), 2.51 (d, J=6.2 Hz, 1H), 3.03 (m, 1H), 3.18 (s, 3H),3.22 (dd, J=14.1, 7.6 Hz, 1H), 3.47 (dd, J=14.3, 6.0 Hz, 1H), 7.10 (dd,J=9.0, 2.8 Hz, 4H), 7.32 (d, J=9.3 Hz, 2H), 7.87 (d, J=9.0 Hz, 2H); ¹³CNMR (126 MHz, CDCl₃) δ 24.2, 26.1, 37.5, 62.5, 118.2, 121.6, 124.0,130.9, 132.6, 145.6, 153.8, 162.1.

Compounds 35b-35g were prepared in the same manner as described for 35a,with the exception corresponding sulfonyl chlorides was used in place ofmesylehloride.

Compound 35b (R₁═R₂═H, R₃=n-Pr, n=1,4-sulfonyloxy). ¹H NMR (300 MHz,CDCl₃) δ 1.14 (t, J=7.5 Hz, 3H), 2.03 (m, 2H), 2.16 (dd, J=5.0, 1.7 Hz,1H), 2.53 (dd, J=6.1, 1.7 Hz, 1H), 3.06 (m, 1H), 3.25 (m, 3H), 3.50 (dd,J=14.4, 5.8 Hz, 1H), 7.13 (d, J=8.8 Hz, 4H), 7.33 (d, J=8.8 Hz, 2H),7.89 (d, J=8.6 Hz, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 12.9, 17.4, 24.2,26.2, 52.2, 62.5, 118.1, 121.6, 124.1, 130.9, 132.6, 145.6, 153.6,162.3.

Compound 35c (R═R₂═H, R₃=i-Pr, n=1,4-sulfonyloxy). ¹H NMR (300 MHz,CDCl₃) δ 1.56 (d, J=6.6 Hz, 6H), 2.14 (dd, J=5.2, 1.7 Hz, 1H), 2.52 (d,J=6.1 Hz, 1H), 3.06 (m, 1H), 3.22 (dd, J=14.1, 7.5 Hz, 1H), 3.50 (m,2H), 7.10 (d, J=8.6 Hz, 4H), 7.31 (d, J=9.1 Hz, 2H), 7.87 (d, J=8.6 Hz,2H); ¹³C NMR (75 MHz, CDCl₃) δ 16.8, 24.2, 26.1, 52.7, 62.5, 118.0,121.5, 124.0, 130.9, 132.5, 145.5, 153.4, 162.3.

Compound 35d (R₁═R₂═H, R₃=Ph, n=1,4-sulfonyloxy). 39-phenylsulfonyl ¹HNMR (500 MHz, CDCl₃) δ 2.16 (dd, J=5.0, 1.6 Hz, 1H), 2.53 (dd, J=6.2,1.4 Hz, 1H), 3.05 (m, 1H), 3.22 (dd, J=14.1, 7.6 Hz, 1H), 3.49 (dd,J=14.5, 5.9 Hz, 1H), 6.98-7.05 (m, 4H), 7.07 (d, J=9.0 Hz, 2H), 7.56 (t,J=7.9 Hz, 2H), 7.69 (t, J=7.6 Hz, 1H), 7.87 (dd, J=8.6, 6.5 Hz, 4H); ¹³CNMR (126 MHz, CDCl₃) δ 24.3, 26.2, 62.7, 118.2, 121.3, 124.4, 128.6,129.4, 131.0, 132.8, 134.6, 135.2, 146.2, 153.7, 162.3.

Compound 35e (R₁═R₂═H, R₃=4-methylphenyl, u=1,4-sulfonyloxy). ¹H NMR(500 MHz, CDCl₃) δ 2.15 (dd, J=5.2, 1.7 Hz, 1H), 2.46 (s, 3H), 2.53 (d,J=6.2 Hz, 1H), 3.06 (m, 11:1), 3.22 (dd, J=14.5, 7.6 Hz, 1H), 3.48 (m,1H), 7.01 (d, J=12.1 Hz, 4H), 7.07 (d, J=9.0 Hz, 2H), 7.34 (d, J=8.3 Hz,2H), 7.73 (d, J=7.9 Hz, 2H), 7.88 (d, J=8.6 Hz, 2H); ¹³C NMR (126 MHz,CDCl₃) δ 22.8, 24.3, 26.2, 62.7, 118.2, 121.3, 124.4, 128.7, 130.0,131.0, 132.3, 132.8, 145.8, 146.4, 153.6, 162.4.

Compound 35f (R₁═R₂═H, R₃=4-methoxyphenyl, n=1,4-sulfonyloxy). ¹H NMR(500 MHz, CDCl₃) δ 2.14 (d, J=5.2 Hz, 1H), 2.52 (d, J=6.2 Hz, 1H), 3.04(m, 1H), 3.21 (dd, J=14.5, 7.6 Hz, 1H), 3.48 (dd, J=14.1, 5.9 Hz, 1H),3.88 (s, 3H), 7.00 (dd, J=11.4, 9.6 Hz, 6H), 7.06 (d, J=8.6 Hz, 2H),7.75 (d, J=8.6 Hz, 2H), 7.87 (d, J=8.6 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃)δ 24.3, 26.2, 55.9, 62.6, 114.5, 118.1, 121.3, 124.4, 124.4, 126.3,130.9, 131.0, 132.7, 146.3, 153.5, 162.3, 164.4.

Compound 35g (R₁═R₂═H, R₃=2,4,6-trimethylphenyl, n=1,4-sulfonyloxy). ¹HNMR (500 MHz, CDCl₃) δ 1.21 (d, J=6.9 Hz, 12H), 1.27 (d, J=7.2 Hz, 6H),2.15 (dd, J=5.2, 1.4 Hz, 1H), 2.53 (m, 1H), 2.94 (m, 1H), 3.05 (m, 1H),3.21 (dd, J=14.1, 7.6 Hz, 1H), 3.49 (dd, J=14.5, 5.9 Hz, 1H), 4.08 (m,1H), 7.01-7.06 (m, 6H), 7.21-7.23 (m, 2H), 7.86 (d, J=9.0 Hz, 2H); ¹³CNMR (126 MHz, CDCl₃) δ 23.7, 24.3, 24.7, 26.2, 29.9, 34.4, 62.7, 117.9,121.5, 124.1, 124.6, 129.4, 130.9, 132.6, 146.3, 151.4, 153.5, 154.7,162.5.

Compound 37b (R₁═R₂═H, R₃=i-Bu, n=1,4-carboxymethyl). To s stirredsolution of 36a (50 mg, 0.14 mmol), EDC (53 mg, 0.28 mmol), DMAP (1.3mg, 0.01 mmol) in CH₂Cl₂ (5 ml) was added iso-butanol (0.013 mml, 10.43mg, 0.14 mmol) and the mixture was stirred at room temperature for 2hours. The solution was concentrated under reduced pressure. The residuewas dissolved in ethyl acetate, washed with 10% citric acid solution,saturated NaHCO₃-solution, water and brine, dried over MgSO₄ andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (hexane/ethyl acetate=2/1) to give 37b as acolorless oil (35 mg, 0.08 mmol, 60%). ¹H-NMR (300 MHz, CDCl₃, TMS): δ0.92 (d, 6H, J=6.7 Hz), 1.94 (sep, 1H, J=6.7 Hz), 2.17 (dd, 1H, J=5.1,1.8 Hz), 2.55 (dd, 1H, J=6.3, 1.7 Hz), 3.07 (m, 1H), 3.18 (dd, 1H,J=14.0, 7.8 Hz), 3.53 (dd, 1H, J=13.9, 5.4 Hz), 3.66 (s, 2H), 3.91 (d,2H, J=6.6 Hz), 7.07 (m, 4H), 7.35 (m, 2H), 7.87 (m, 2H). ¹³C—N (500 MHz,CDCl₃, TMS): δ 19.0, 24.3, 26.1, 27.7, 40.7, 62.7, 71.1, 117.4, 120.6,130.7 (d), 131.2 (d), 131.3, 131.9, 153.7, 162.9, 171.4 MS (FAB) m/z420.1046 (calcd for C₂₁H₂₄O₅S₂ [M]⁺ 420.1065) TLC R_(f)=0.54 (1:1hexanes/EtOAc)

Compounds 37c-37g were prepared in the same manner as described forcompound 37b with the exception of corresponding alcohol was usedinstead of isobutanol.

Compound 37c (R₁═R₂═H, R₃=4-chlorobenzyl, n=1,4-carboxymethyl). ¹H-NMR(300 MHz, CDCl₃, TMS): δ 2.16 (dd, 1H, 5.1, 1.7 Hz), 2.54 (dd, 1H, 6,1.4 Hz), 3.06 (m, 1H), 3.18 (dd, 1H, 14.1, 7.8 Hz), 3.52 (dd, 1H, 13.8,5.4 Hz), 3.69 (s, 2H), 5.12 (s, 2H), 7.06 (m, 4H), 7.29 (m, 6H), 7.86(m, 2H) ¹³C-NMR (500 MHz, CDCl₃, TMS): δ 24.3, 26.1, 40.5, 62.7, 66.0,117.4, 117.8, 120.6, 128.8, 129.6, 130.7, 130.8, 131.2 (d), 132.0,134.2, 153.9, 162.8, 171.1 MS (FAB) m/z 489.0576 (calcd for C₂₄H₂₂ClO₅[M+H]⁺ 489.0597) TLC R_(f)=0.53 (1:1 hexanes/EtOAc)

Compound 37d (R₁═R₂═H, R₃=thiophen-2-yl, n=1,4-carboxymethyl). ¹H-NMR(500 MHz, acetone, TMS): δ 2.18 (dd, 1H, J=5.0, 1.4 Hz), 2.55 (dd, H,6.0, 1.3 Hz), 3.06 (m, 1H), 3.40 (dd, 1H, 14.5, 7.3 Hz), 3.59 (dd, 1H,14.5, 6.1 Hz), 3.74 (s, 2H), 5.33 (s, 2H), 7.01 (m, 1H), 7.14 (m, 5H),7.41 (m, 2H), 7.48 (m, 1H), 7.94 (m, 2H) ¹³C-NMR (500 MHz, CDCl₃, TMS):δ 24.3, 26.1, 40.4, 61.1, 62.7, 117.4, 117.7, 120.6, 126.9, 127.0,128.3, 130.7, 130.8 (d), 131.2 (d), 131.9, 137.6, 153.9, 162.9, 171.1 MS(FAB)—(measurement was not possible) TLC R_(f)=0.53 (1:1 hexanes/EtOAc)

Compound 37e (R₁═R₂═H, R₃=pyridin-2-yl)methyl, n=1,4-carboxymethyl).¹H-NMR (300 MHz, CDCl₃, TMS): δ 2.17 (dd, 1H, J=5.1, 1.7 Hz), 2.54 (dd,1H, J=6.0, 1.4 Hz), 3.07 (m, 1H), 3.19 (dd, 1H, J=13.8, 7.8 Hz), 3.53(dd, 1H, J=14.1, 5.4 Hz), 3.78 (s, 1H), 5.30 (s, 1H), 7.08 (m, 4H), 7.32(m, 4H), 7.72 (d of t, 1H, J=7.8, 1.5 Hz), 7.87 (m, 2H), 8.62 (d, 1H,J=4.9 Hz), ¹³C-NMR (500 MHz, CDCl₃, TMS): δ 24.3, 26.1, 40.4, 62.6,67.3, 117.4, 117.8, 120.6, 121.9, 123.1, 130.8 (t), 131.3 (d), 132.0,136.9, 149.5, 153.9, 155.4, 162.9, 171.1 MS (FAB) m/z 456.0943 (calcdfor C₂₃H₂₂O₅NS₂ [M+H]⁺ 456.0939) TLC R_(f)=0.19 (1:1 hexanes/EtOAc)

Compound 37f (R₁═R₂═H, R₃— pyridin-3-yl)methyl, n=1,4-carboxymethyl).¹H-NMR (500 MHz, acetone, TMS): δ 2.18 (dd, 1H, J=5.0, 1.4 Hz), 2.55(dd, 1H, J=6.5, 1.2 Hz), 3.06 (quinett t,t, 1H, J=6.2, 1.1 Hz), 3.41(dd, 1H, J=14.5, 7.3 Hz), 3.59 (dd, 1H, J-14.5, 6.1 Hz), 3.79 (s, 2H),5.21 (s, 2H), 7.15 (m, 4H), 7.37 (ddd, 1H, J=5.0, 8.0, 0.8 Hz), 7.43 (m,2H), 7.77 (m, 1H), 7.95 (m, 2H), 8.54 (dd, 1H, J=4.5, 1.6 Hz), 8.59 (d,1H, J=1.8 Hz) ¹³C-NMR (500 MHz, CDCl₃, TMS): δ 24.3, 26.1, 40.4, 62.6,64.2, 117.5, 117.8, 120.6, 123.6, 130.5, 130.8 (d), 131.2 (d), 132.0,136.2, 149.4, 149.5, 154.0, 162.8, 171.1 MS (FAB) m/z 456.0961 (calcdfor C₂₃H₂₂O₅NS₂ [M+H]⁺ 456.0939) TLC R_(f)=0.14 (1:1 hexanes/EtOAc)

Compound 37g (R₁═R₂═H, R₃=benzyl, n=1; 4-carboxymethyl). ¹H-NMR (300MHz, CDCl₃, TMS): δ 2.17 (dd, 1H, J=5.1, 1.7 Hz), 2.55 (dd, 1H, J=6.0,1.1 Hz), 3.07 (m, 1H), 3.19 (dd, 1H, I=14.1, 7.9 Hz), 3.53 (dd, 1H, J14.1, 5.4 Hz), 3.71 (s, 1H), 5.17 (s, 1H), 7.08 (m, 4H), 7.35 (m, 61),7.87 (m, 2H) ¹³C-NMR (500 MHz, CDCl₃, TMS): δ 24.2, 26.1, 40.6, 62.7,66.8, 117.7, 120.6, 128.2, 128.4, 128.6, 130.7, 130.8, 130.9, 131.2,132.0, 135.7, 153.9, 162.9, 171.2 MS (FAB) m/z 455.0989 (calcd forC₂₄H₂₃O₅S₂ [M+H]⁺ 455.0987) TLC R_(f)=0.53 (1:1 hexanes/EtOAc)

Compound 37h (R₁═R₂═H, R₃=perfluorophenyl, n=1,4-carboxymethyl). To astirred solution of 36a (521 mg, 1.43 mmol), EDC (548 mg, 2.86 mmol),DMAP (12 mg, 0.1 mmol) in CH₂Cl₂ (30 ml) was added pentafluorophenol(PfP) (263 mg, 1.43 mmol) and the mixture was stirred overnight at roomtemperature. The solution was concentrated under reduced pressure. Theresidue was dissolved in ethyl acetate, washed with 10% citric acidsolution, saturated NaHCO₃-solution, water and brine, dried over MgSO₄and concentrated under reduced pressure to give the crude PfP-ester (636mg). The crude material was used for the next step.

Compound 38a (R₁═R₂═H, R₃=diethyl, n=1,4-acetamide). To a stirredsolution of 37h (54 mg, 0.10 mmol) in CH₂Cl₂ (5 ml) is addeddiethylamine (14.6 mg, 0.10 mmol) and the mixture is stirred overnightat room temperature. The solution was concentrated under reducedpressure. The residue was dissolved in ethyl acetate, washed with 10%citric acid solution, saturated NaHCO₃ solution, water and brine, driedover MgSO₄ and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (hexane/ethyl acetate=1/2)to give 38a (23 mg, 0.055 mmol, 55%) as a colorless oil. ¹H-NMR (500MHz, CDCl₃, TMS): δ 1.14 (t, 3H, J=7.1 Hz), 1.16 (t, 3H, J=7.1 Hz) 2.16(dd, 1H, J=5.0, 1.8 Hz), 2.54 (dd, 1H, J=6.0, 1.1 Hz), 3.06 (m, 1H),3.17 (dd, 1H, 14.0, 7.9 Hz), 3.36 (q, 2H, J=7.1 Hz), 3.41 (q, 2H, J=7.1Hz), 3.52 (dd, 1H, J=14.0, 5.5 Hz), 3.71 (s, 2H), 7.06 (m, 4H), 7.31 (m,2H), 7.85 (m, 2H) ¹³C-NMR (500 MHz, CDCl₃, TMS): δ 13.0, 14.4, 24.3,26.1, 39.8, 40.4, 42.4, 62.6, 117.3, 117.6, 120.6, 130.7 (d), 130.8 (d),131.8, 132.7, 153.4, 163.0, 169.8 MS (FAB) m/z 420.1306 (calcd forC₂₁H₂₆O₄NS₂ [M+H]⁺ 420.1303) TLC R_(f)=0.30 (1:2 hexanes/EtOAc)

Compounds 38b-38e were prepared in the same manner as described forcompound 38a with the exception of corresponding amine was used insteadof diethylamine.

Compound 38b (R₁═R₂═H, R₃=benzyl, n=1,4-acetamide). ¹H-NMR (300 MHz,CDCl₃, TMS): δ 2.17 (dd, 1H, J=5.1, 1.8 Hz), 2.55 (dd, 1H, J=6.0, 1.7Hz), 3.07 (m, 1H), 3.19 (dd, 1H, J=14.0, 7.8 Hz), 3.52 (dd, 1H, J=14.1,5.7 Hz), 3.63 (s, 2H), 4.46 (d, 2H, J=5.7 Hz), 5.80 (m, 1H), 7.08 (m,4H), 7.29 (m, 7H), 7.87 (m, 2H) ¹³C-NMR (500 MHz, CDCl₃, TMS): δ 24.2,26.1, 43.0, 43.8, 62.6, 117.5, 117.8, 117.9, 120.8, 127.7, 128.8, 130.8(d), 131.2 (d), 131.8, 132.1, 138.0, 154.1, 162.7, 170.4 MS (FAB) m/z454.1162 (calcd for C₂₄H₂₄O₄NS₂ [M+2H]⁺ 454.1147) TLC R_(f)=0.30 (1:1hexanes/EtOAc)

Compound 38c (R₁═R₂═H, R₃=(furan-2-yl)methyl, n=1,4-acetamide). ¹H-NMR(300 MHz, CDCl₃) δ 2.17 (dd, 1H, J=4.8, 1.8 Hz), 2.55 (dd, 1H, J=6.0,1.7 Hz), 3.07 (m, 1H), 3.20 (dd, 1H, J=13.8, 7.7 Hz), 3.52 (dd, 1H,J=14.1, 5.6 Hz), 3.61 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 5.80 (m, 1H),6.20 (d, 1H, J=3.3 Hz), 6.32 (m, 1H), 7.09 (m, 4H), 7.33 (m, 3H), 7.87(m, 2H) ¹³C-NMR (500 MHz, CDCl₃, TMS): δ 24.2, 26.1, 36.7, 42.8, 62.7,107.5, 110.4, 117.5, 117.8, 120.8, 130.8 (d), 131.2 (d), 131.6, 132.1,142.3, 150.9, 154.1, 162.7, 170.3 MS (FAB) m/z 444.0920 (calcd forC₂₂H₂₂O₅NS₂ [M+2H]⁺ 444.0939) TLC R_(f)=0.32 (1:1 hexanes/EtOAc)

Compound 38d (R₁═R₂═H, R₃=(pyridin-2-yl)methyl, n=1,4-acetamide). ¹H-NMR(500 MHz, CDCl₃): δ 2.16 (dd 1H, J=5.0, 1.8 Hz), 2.54 (dd 1H, J=6.0, 1.5Hz), 3.06 (m, 1H), 3.18 (dd, 1H, J=14.5, 7.8 Hz), 3.52 (dd, 1H, J=14.5,5.6 Hz), 3.66 (s, 2H), 4.59 (d, 2H, J=5.0 Hz), 7.07 (m, 4H), 7.32 (m,4H), 7.80 (m, 3H), 8.51 (d, 1H, J=5.0 Hz) ¹³C-NMR (500 MHz, CDCl₃): δ24.2, 26.1, 42.8, 44.1, 62.7, 117.4, 117.8 (d), 120.8, 122.8, 122.9,130.8, 131.3 (d), 131.9 (d), 137.9, 148.0, 153.9, 155.7, 162.9, 170.8 MS(FAB) m/z 455.1100 (calcd for C₂₃H₂₃O₄N₂S₂ [M+2H]⁺) TLC R_(f)=0.42 (30:1CH₂Cl₂/MeOH).

Compound 38e (R₁═R₂═H, R₃=(pyridin-3-yl)methyl, n=1,4-acetamide). ¹H-NMR(500 MHz, CDCl₃): δ 2.16 (dd, 1H, J=5.0, 1.7 Hz), 2.54 (dd, 1H, J=6.0,1.8 Hz), 3.06 (m, 1H), 3.19 (dd, 1H, J=14.5, 7.7 Hz), 3.50 (dd, 1H,J=14.0, 5.7 Hz), 3.63 (s, 2H), 4.46 (d, 2H, J=6.06 Hz), 5.98 (m, 1H),7.07 (m, 4H), 7.30 (m, 3H), 7.60 (m, 1H), 7.83 (m, 2H), 8.47 (d, 1H,J=2.1 Hz), 8.51 (dd, 1H, J=5.0, 1.5 Hz) ¹³C-NMR (500 MHz, CDCl₃): δ24.2, 26.1, 41.2, 42.8, 62.6, 117.6, 117.9 (d), 120.8, 123.7, 130.8 (d),131.2 (d), 131.5, 132.2, 133.9, 135.7, 148.8, 154.2, 162.6, 170.7 MS(FAB) m/z 455.1101 (calcd for C₂₃H₂₃O₄N₂S₂ [M+2H]⁺ 455.1099) TLCR_(f)=0.40 (30:1 CH₂Cl₂/MeOH).

Compounds of Scheme 11.

Compound 40=compound 28a (R₂=p-NO₂, R₂=allylthio).

Compound 42a (R═SO₂CH₃). To a stirred solution of 40 (636 mg, 2.21 mmol)in THF (22 mL) were added acetic acid (2.54 mL, 44.2 mmol) and zincpowder (5.80 g, 88.4 mmol) at room temperature, and the suspension wasstirred for 30 minutes (an exothermic reaction). After dilution withethyl acetate, the mixture was filtered through Celite. The filtrate waswashed with saturated NaHCO₃ and brine, dried over Na₂SO₄, andconcentrated under reduced pressure to give a crude 41 (577 mg) as anorange oil, which was employed in the next reaction withoutpurification. To a stirred solution of 41 (577 mg) in CH₂Cl₂ (10 ml)were added pyridine (894 μL, 11.1 mmol) and methanesulfonyl chloride(205 μL, 2.65 mmol) at ice-water temperature. After 15 minutes, themixture was warmed to room temperature and the stirring was continuedfor an additional 2 hours. Subsequent to the addition of saturatedNaHCO₃, the mixture was extracted with ethyl acetate (3×). The combinedorganic layer was washed with 1 M aqueous HCl, saturated NaHCO₃ solutionand brine, dried over Na₂SO₄, and concentrated under reduced pressure.

The resultant residue was purified by silica gel column chromatography(CH₂Cl₂) to give 42a (662 mg, 89% from 40) as a pale red solid. Compound41: ¹H NMR (300 MHz, CDCl₃): δ 3.45 (br.d, 2H, J=7.2 Hz), 3.59 (br.s,2H), 5.01-5.06 (m, 2H), 5.84 (ddt, 1H, J=17.1, 9.6, 6.9 Hz), 6.66-6.70(m, 2H), 6.83-6.88 (m, 4H), 7.29-7.32 (m, 2H). Compound 42: 1H MR (300MHz, CDCl₃): δ 3.01 (s, 3H), 3.50 (dt, 2H, J=7.2, 1.2 Hz), 5.04-5.11 (m,2H), 5.86 (ddt, 1H, J=16.8, 10.2, 6.9 Hz), 6.67 (br.s, 1H), 6.90-6.95(m, 2H), 6.96-7.01 (m, 2H), 7.20-7.26 (m, 2H), 7.32-7.37 (m, 2H); ¹³CNMR (75 MHz, CDCl₃): δ 38.4, 39.3, 117.5, 119.3, 119.9, 123.8, 130.2,132.0, 132.9, 133.8, 155.2, 156.1; HRMS (FAB) calcd for C₁₆H₁₇NO₃S₂ (M⁺)335.0650, found 335.0639.

Compound 42b (R═COCH₃). To a stirred solution of 41 (794 mg), which wasprepared from 40 (830 mg, 2.89 mmol) in the same manner as described forcompound 42, in CH₂Cl₂ (15 ml) were added pyridine (500 μL, 6.18 mmol)and acetic anhydride (292 μL, 3.09 mmol) at ice-water temperature, andthe mixture was stirred at the same temperature for 1 hour. Subsequentto the addition of saturated NaHCO₃, the mixture was extracted withethyl acetate (3×). The combined organic layer was washed with 1 Maqueous HCl, saturated NaHCO₃ solution and brine, dried over Na₂SO₄, andconcentrated under reduced pressure.

The resultant residue was purified by silica gel column chromatography(ethyl acetate/CH₂Cl₂=1/8) to give 42b (782 mg, 99% from 40) as a whitesolid. ¹H NMR (500 MHz, CDCl₃): δ 2.17 (s, 3H), 3.47 (d, 2H, J=7.0 Hz),5.04-5.07 (m, 2H), 5.85 (ddt, 1H, J=17.0, 10.0, 7.0 Hz), 6.87-6.90 (m,2H), 6.94-6.97 (m, 2H), 7.31-7.35 (m, 2H), 7.44-7.47 (m, 2H), 7.54(br.s, 1H); ¹³C NMR (75 MHz, CDCl₃): δ 24.4, 38.5, 117.5, 118.7, 119.6,121.7, 129.0, 132.9, 133.6, 133.7, 153.1, 156.7, 168.4; HRMS (FAB) calcdfor C₁₇H₁₇NO₂S (M⁺) 299.0980, found 299.0980.

Conversion from compounds 40, 42a, and 42b to compounds 4f, 43-44respectively was followed by the same method as described in Scheme 6.

Compound 4f (R₁=4-NO₂, R₂═H). ¹H NMR (500 MHz, CDCl₃): δ 2.51 (dd, 1H,J=4.5, 2.5 Hz), 2.83 (t, 1H, J=4.5 Hz), 3.28 (dd, 1H, J=14.0, 7.0 Hz),3.35 (m, 1H), 3.41 (dd, 1H, J=14.0, 4.0 Hz), 7.16-7.17 (m, 2H),7.23-7.25 (m, 2H), 7.99-8.01 (m, 2H), 8.28-8.30 (m, 2H); ¹³C NMR (125MHz, CDCl₃): δ 45.7, 45.8, 59.6, 119.1, 119.6, 126.2, 131.0, 134.9,144.0, 160.2, 160.8; HRMS (FAB) calcd for C₁₅H₁₄NO₆S (M+H⁺) 336.0542,found 336.0545.

Compound 5f (R₁=4-NO₂, R₂=H). ¹H NMR (500 MHz, CDCl₃): δ 2.58 (dd, 1H,J=6.0, 2.0 Hz), 3.10 (m, 1H), 3.31 (dd, 1H, J=14.0, 7.5 Hz), 3.52 (dd,1H, J=14.0, 6.5 Hz), 7.15-7.18 (m, 2H), 7.23-7.26 (m, 2H), 7.97-8.00 (m,2H), 8.28-8.31 (m, 2H); ¹³C NMR (125 MHz, CDCl₃): δ 24.0, 26.0, 62.5,119.1, 119.8, 126.2, 131.2, 134.4, 160.3, 160.8; ¹³C NMR (125 MHz,acetone-d₆): δ 24.5, 27.2, 62.6, 120.2, 121.0, 127.1, 132.3, 136.2,145.0, 161.1, 162.4; HRMS (FAB) calcd for C₁₅H₁₄NO₅S₂ (M⁺H⁺) 352.0313,found 352.0297.

Compound 4g=compound 43a (R₁=4-CH₃SO₂NH, R₂=H). ¹H NMR (300 MHz, CDCl₃):δ 2.49 (dd, 1H, J=5.1, 1.8 Hz), 2.83 (m, 1H), 3.06 (s, 3H), 3.27-3.36(m, 3H), 6.77 (br.s, 1H), 7.08-7.11 (m, 4H), 7.28-7.33 (m, 2H),7.88-7.93 (m, 2H); ¹³C NMR (125 MHz, acetone-d₆): δ 39.4, 45.9, 46.6,59.9, 118.3, 122.3, 123.6, 131.6, 134.6, 136.5, 152.7, 163.5; HRMS (FAB)calcd for C₁₆H₁₇NO₆S₂ (M⁺) 383.0497, found 383.0496.

Compound 5g=compound 44a (R₁=4-CH₃SO₂NH, R₂=H). ¹H NMR (300 MHz, CDCl₃):δ 2.17 (dd, 1H, J=5.1, 1.8 Hz), 2.55 (dd, 1H, J=6.3, 1.2 Hz), 3.06 (s,3H), 3.07 (m, 1H), 3.22 (dd, 1H, J=14.1, 7.8 Hz), 3.50 (dd, 1H, J=14.1,5.7 Hz), 6.72 (br.s, 1H), 7.08-7.12 (m, 4H), 7.30-7.33 (m, 2H),7.87-7.91 (m, H); ¹³C NMR (125 MHz, acetone-d₆): δ 24.4, 27.2, 39.3,62.6, 118.4, 122.3, 123.6, 131.9, 133.9, 136.4, 152.7, 163.6; HRMS (FAB)calcd for C₁₆H₁₇NO₅S₃ (M⁺) 399.0269, found 399.0268.

Compound 4h=compound 43b (R₁=4-CH₃CONH, R₂=H). ¹H NMR (300 MHz, CDCl₃):δ 2.20 (s, 3H), 2.48 (dd, 1H, J=4.5, 1.5 Hz), 2.82 (m, 1H), 3.26-3.34(m, 3H), 7.03-7.08 (m, 4H), 7.41 (br.s, 1H), 7.55-7.58 (m, 2H),7.85-7.88 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 24.1, 45.7, 45.8, 59.8,117.6, 120.8, 122.0, 130.4, 133.0, 135.4, 151.1, 163.0, 168.5; HRMS(FAB) calcd for C₁₇H₁₈NO₅S (M⁺H⁺) 348.0906, found 348.0913.

Compound 5h=compound 44b (R₁=4-CH₃CONH, R₂=H). ¹H NMR (300 MHz, CDCl₃):δ 2.16 (dd, 1H, J=5.1, 1.8 Hz), 2.20 (s, 3H), 2.54 (dd, 1H, J=6.3, 1.5Hz), 3.06 (m, 1H), 3.19 (dd, 1H, J=14.1, 7.8 Hz), 3.52 (dd, 1H, J=14.1,5.7 Hz), 7.03-7.08 (m, 4H), 7.52 (br.s, 1H), 7.56-7.59 (m, 2H),7.84-7.87 (m, 2H); ¹³C NMR (125 MHz, CDCl₃): δ 24.1, 24.4, 26.0, 62.6,117.4, 121.0, 121.8, 130.7, 131.6, 135.2, 150.7, 163.1, 168.6; HRMS(FAB) calcd for C₁₇H₁₈NO₄S₂ (M⁺H⁺) 364.0677, found 364.0651

Conversion from compound 29a to compounds 3i-5i was followed by the samemethod as described in Schemes 8 and 11.

Compound 21b (R₁=4-CH₃SO₂NH, 2-pyridyl, R₂═H). ¹H NMR (500 MHz, CDCl₃) δ2.96 (s, 3H), 3.03 (br.s., 3H), 3.10 (br.s., 3H), 6.96 (d, J=9.0 Hz,1H), 7.12 (d, J=8.8 Hz, 2H), 7.51 (d, J=8.8 Hz, 2H), 7.77 (dd, J=8.8,2.8 Hz, 1H), 7.99 (d, J=2.8 Hz, 1H); HRMS calcd for C₂₅H₃₀N₅O₆S (M+H⁺)528.1917, found 528.1920.

Compound 4i (R₁=4-CH₃SO₂NH, 2-pyridyl, R₂═H). ¹H NMR (500 MHz, 5% CD₃ODin CD₂Cl₂) δ 2.46 (dd, J=4.6, 2.0 Hz, 1H), 2.79 (t, J=4.4 Hz, 1H), 2.96(s, 3H), 3.24-3.40 (m, 3H), 7.03 (d, J=8.8 Hz, 1H), 7.29 (d, J=8.4 Hz,2H), 7.79 (dd, J=8.8, 2.8 Hz, 1H), 7.93 (d, J=8.6 Hz, 2H), 8.05 (d,J=3.0 Hz, 1H); ¹³C NMR (126 MHz, 5% CD₃OD in CD₂Cl₂) δ 39.7, 46.2, 46.3,60.1, 113.9, 121.3, 130.8, 132.0, 134.7, 134.9, 141.1, 159.9, 160.0 (s);HRMS calcd for C₁₅H₁₇N₂O₆S₂ (M+H⁺) 385.0528, found 385.0531.

Compound 5i (R₁=4-CH₃SO₂NH, 2-pyridyl, R₂═H). ¹H NMR (500 MHz, 5% CD₃ODin CD₂Cl₂) δ 2.16 (dd, J=5.2, 1.8 Hz, 1H), 2.53 (dd, J=6.6, 1.8 Hz, 1H),2.97 (s, 3H), 3.06 (m, 1H), 3.25 (dd, J=14.4, 7.6 Hz, 1H), 3.52 (dd,J=14.4, 6.0 Hz, 1H), 7.04 (dd, J=8.8, 0.4 Hz, 1H), 7.31 (d, J=8.6 Hz,2H), 7.79 (dd, J=8.8, 2.8 Hz, 1H), 7.92 (d, J=8.8 Hz, 2H), 8.04 (d,J=2.8 Hz, 1H); ¹³C NMR (126 MHz, 5% CD₃OD in CD₂Cl₂) δ 24.5, 26.5, 39.8,63.0, 113.8, 121.4, 131.0, 131.9, 134.4, 134.7, 141.1, 159.9, 160.1 (s);HRMS calcd for C₁₅H₁₇N₂O₅S₃ (M+H) 401.0300, found 401.0290.

Compound 48. 2-(4-Bromophenoxy)-phenol was prepared by reported method.A mixture of phenol (2.65 g, 10.0 mmol) and K₂CO₃ (1.45 g, 10.5 mmol) inDMF (20 mL) was stirred for 0.5 hours, and then benzylbromide (1.56 mL,ml3.0 mmol) was added. The reaction mixture was stirred at roomtemperature for 3 hours, then was diluted with ethyl acetate and water.The layers were separated and the aqueous layer was extracted with ethylacetate. The combined organic layer was washed with water, 2 N HCl,sat'd NaHCO₃, and brine, dried under anhydrous MgSO₄, filtered, anddried under reduced pressure. The residue was purified by columnchromatography to give the desired product as a white solid (3.00 g,84%). ¹H NMR (500 MHz, CDCl₃) δ 5.06-5.13 (m, 2H), 6.85 (d, J=9.0 Hz,2H), 7.00 (t, J=9.0 Hz, 1H), 7.05-7.23 (m, 5H), 7.26-7.37 (m, 3H), 7.42(d, J=9.0 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 70.6, 114.6, 115.3, 118.6,121.9, 122.4, 125.7, 127.2, 128.0, 128.6, 132.5, 136.8, 144.9, 150.7,157.9;

Conversion from compound 48 to compounds 52a-55a was followed by thesame method as described in Scheme 5.

Compound 2j=compound 52a (R₁=2-benzyloxy, R₂═H). ¹H NMR (500 MHz, CDCl₃)δ 3.07 (ddd, J=44.7, 13.8, 5.4 Hz, 2H), 3.68 (ddd, J=19.9, 11.2, 4.4 Hz,1H), 3.83-3.95 (m, 1H), 5.10 (s, 2H), 6.93 (d, J=8.6 Hz, 2H), 7.02 (t,J=7.6 Hz, 1H), 7.05-7.25 (m, 5H), 7.27-7.36 (m, 3H), 7.41 (d, J=8.6 Hz,2H); ¹³C NMR (126 MHz, CDCl₃) δ 40.1, 48.1, 69.5, 70.8, 115.3, 117.5,121.9, 122.4, 125.7, 126.6, 127.1, 127.9, 128.5, 132.1, 133.4, 136.8,144.7, 150.7, 158.5.

Compound 3j (R₁=2-benzyloxy, R₂═H). ¹H NMR (500 MHz, CDCl₃) δ 2.43 (dd,J=4.8, 2.4 Hz, 1H), 2.78 (t, J=4.4 Hz, 1H), 3.22 (dd, J=14.0, 5.2 Hz,1H), 3.26-3.30 (m, 1H), 5.04-5.08 (m, 2H), 7.03-7.08 (m, 3H), 7.09-7.21(m, 4H), 7.22-7.33 (m, 4H), 7.87 (d, J=8.8 Hz, 2H); ¹³C NMR (126 MHz,CDCl₃) δ 46.0, 46.1, 59.8, 70.7, 115.1, 116.5, 122.0, 123.1, 126.8,127.1, 128.1, 128.6, 130.4, 131.9, 136.4, 143.2, 150.7, 163.4;

Compound 4j=compound 53a (R₁=2-benzyloxy, R₂═H). ¹H NMR (500 MHz, CDCl₃)δ 2.47 (dd, J=5.0, 2.6 Hz, 1H), 2.76 (t, J=3.8 Hz, 1H), 2.88 (dd,J=14.0, 6.2 Hz, 1H), 3.12 (dd, J=13.6, 5.6 Hz, 1H), 3.16-3.20 (m, 1H),5.09-5.12 (m, 2H), 6.95 (d, J=9.0 Hz, 2H), 7.03 (td, J=7.6, 1.6 Hz, 6H),7.08-7.25 (m, 5H), 7.47 (d, J=8.8 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ38.2, 47.4, 51.1, 70.6, 115.1, 117.2, 121.7, 122.3, 125.5, 127.0, 127.2,127.8, 128.4, 133.7, 136.7, 144.6, 150.6, 158.2;

Compound 4jj=compound 54a (R₁=2-hydroxy, R₂═H). Compound 4j (0.50 g, 1.3mmol) was stirred in the presence of Pd(OH)₂ (0.15 g) inethylacetate/i-PrOH (20 mL) for 0.5 h in the atmosphere of H2. Thereaction mixture was filtered through a layer of celite and the filtratewas concentrated under reduced pressure. The residue was purified bycolumn chromatography to give the desired product (0.28 g, 70%). ¹H NMR(500 MHz, CDCl₃) δ 2.45 (dd, J=3.4, 1.8 Hz, 1H), 2.77-2.83 (m, 1H),3.24-3.30 (m, 3H), 6.89-6.95 (m, 1H), 6.97-7.02 (m, 1H), 7.04-7.10 (m,3H), 7.11-7.18 (m, 1H), 7.82 (d, J=9.0 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃)δ 46.0, 59.7, 117.2, 117.5, 121.3, 126.9, 130.7, 132.7, 141.4, 148.2,162.5;

Compound 5j (R₁=2-benzyloxy, R₂═H). ¹H NMR (500 MHz, CDCl₃) δ 2.10 (dd,J=5.0, 1.6 Hz, 1H), 2.49 (d, J=6.2 Hz, 1H), 3.04 (m, 1H), 3.13 (dd,J=14.1, 8.1 Hz, 1H), 3.55 (dd, J=14.2, 5.2 Hz, 1H), 5.07 (s, 2H),7.04-7.09 (m, 3H), 7.11-7.17 (m, 3H), 7.23-7.31 (m, 5H), 7.85 (d, J=8.8Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ24.5, 26.3, 62.8, 70.7, 115.1, 116.6,122.1, 123.0, 126.9, 127.1, 128.2, 128.6, 130.6, 131.4, 136.4, 143.2,150.7, 163.5.

Compound 5jj=compound 55a (R₁=2-hydroxy, R₂═II). ¹H NMR (500 MHz, CDCl₃)δ 2.15 (dd, J=5.0, 1.6 Hz, 1H), 2.53 (dd, J=6.0, 1.4 Hz, 8H), 3.01-3.08(m, 1H), 3.18 (dd, J=14.2, 7.8 Hz, 1H), 3.50 (dd, J=14.4, 5.8 Hz, 1H),5.78-5.85 (m, 1H), 6.94 (td, J=8.0, 1.6 Hz, 1H), 7.00 (dd, J=8.0, 1.2Hz, 1H), 7.06-7.20 (m, 4H), 7.83 (d, J=8.8 Hz, 2H); ¹³C NMR (126 MHz,CDCl₃) δ 24.4, 26.2, 62.8, 117.5, 117.5, 121.1, 121.4, 126.9, 130.9,132.4, 141.5, 148.1, 162.4.

Compounds of Scheme 12.

Compound 51. ¹H NMR (500 MHz, CDCl₃) δ 5.06-5.08 (m, 2H), 6.85 (d, J=9.0Hz, 2H), 6.97-7.00 (m, 4H), 7.34-7.50 (m, 7H); ¹³C NMR (126 MHz, CDCl₃)δ 70.7, 115.0, 116.2, 119.5, 121.0, 127.7, 128.2, 128.8, 132.7, 137.1,150.0, 155.5, 157.9.

Conversion from compound 51 to compounds 52b-55b was followed by thesame method as described in Scheme 5.

Compound 2k=compound 52b (R═4-benzyloxy, R₂═H). ¹H NMR (500 MHz, CDCl₃)δ 3.05 (ddd, J=44.5, 13.8, 5.5 Hz, 2H), 3.67 (ddd, J=17.6, 11.0, 4.5 Hz,2H), 5.06 (s, 2H), 6.90 (d, J=8.6 Hz, 2H), 6.98 (s, 4H), 7.34-7.47 (m,7H); ¹³C NMR (126 MHz, CDCl₃) δ 39.9, 48.1, 69.6, 70.7, 116.2, 118.4,121.2, 127.2, 127.6, 128.2, 128.8, 133.4, 137.1, 149.9, 155.5, 158.5.

Compound 3k (R₁=4-benzyloxy, R₂═H). ¹H NMR (500 MHz, CDCl₃) δ 2.49 (dd,J=4.8, 2.8 Hz, 1H), 2.79 (t, J=4.5 Hz, 1H), 2.88 (dd, J=13.8, 5.9 Hz,1H), 3.10 (dd, J=13.8, 4.8 Hz, 1H), 3.15-3.21 (m, 1H), 5.08 (s, 2H),6.92 (d, J=8.6 Hz, 4H), 7.00 (s, 2H), 7.34-7.49 (m, 8H); ¹³C NMR (126MHz, CDCl₃) δ 38.2, 47.5, 51.2, 70.6, 116.1, 118.2, 121.1, 127.6, 127.7,128.1, 128.7, 133.8, 137.0, 149.9, 158.3.

Compound 4k=compound 53b (R₁=4-benzyloxy, R₂═H). ¹H NMR (500 MHz, CDCl₃)δ 2.48 (dd, J=5.0, 1.9 Hz, 1H), 2.82 (dd, J=6.2, 2.8 Hz, 1H), 3.22-3.37(m, 3H), 5.09 (s, 2H), 7.02-7.09 (m, 4H), 7.33-7.38 (m, 1H), 7.40-7.43(m, 9H), 7.45-7.47 (m, 9H), 7.87 (d, J=9.0 Hz, 2H); ¹³C NMR (126 MHz,CDCl₃) δ 46.0, 59.8, 70.6, 116.4, 117.1, 122.0, 127.6, 128.3, 128.8,130.6, 132.1, 136.8, 148.2, 156.4, 163.8.

Compound 4kk=compound 54b (R₁=4-hydroxy, R₂═H). ¹H NMR (500 MHz, CDCl₃)δ 2.47 (dd, J=5.5, 1.4 Hz, 1H), 2.81 (dd, J=4.5, 2.4 Hz, 1H), 3.23-3.35(m, 3H), 6.86 (d, J=9.0 Hz, 2H), 6.92 (d, J=9.0 Hz, 2H), 7.02 (d, J=9.0Hz, 2H), 7.82 (d, J=9.0 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 46.0, 59.8,116.8, 117.0, 122.1, 130.6, 131.6, 147.3, 154.2, 163.6.

Compound 5k (R₁=4-benzyloxy, R₂═H). ¹H NMR (500 MHz, CDCl₃) δ 2.18 (dd,J=5.2, 1.7 Hz, 1H), 2.56 (dd, J=6.2, 1.4 Hz, 1H), 3.08 (m, 1H), 3.19(dd, J=14.1, 7.9 Hz, 1H), 3.54 (dd, J=14.1, 5.5 Hz, 1H), 5.11 (s, 2H),7.06 (s, 5H), 7.08 (d, J=9.0 Hz, 2H), 7.35-7.51 (m, 4H), 7.87 (d, J=9.0Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 24.4, 26.3, 62.8, 70.7, 116.4,117.2, 121.9, 127.6, 128.2, 128.8, 130.8, 131.6, 136.8, 148.2, 156.4,163.8.

Compound 5kk=compound 55b (R₁=4-hydroxy, R₂═H). ¹H NMR (500 MHz, CDCl₃)δ 2.17 (dd, J=5.2, 1.7 Hz, 1H), 2.55 (dd, J=6.2, 1.7 Hz, 1H), 3.02-3.11(m, 1H), 3.20 (dd, J=14.5, 7.9 Hz, 1H), 3.54 (dd, J=14.5, 5.9 Hz, 1H),6.90 (d, J=8.6 Hz, 2H), 6.97 (d, J=9.0 Hz, 2H), 7.06 (d, J=9.0 Hz, 2H),7.88 (d, J=9.0 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 24.4, 26.2, 62.8,117.0, 117.2, 122.2, 130.8, 131.2, 147.9, 153.5, 164.0.

Compounds of Scheme 13.

Synthesis of compounds 57 (=compound 21a), 58 (compound 3d) is describedin Scheme 8. Conversion from compound 58 to compounds 59-61 was followedby the same method as described in Schemes 5 and 12.

Compound 4d=compound 59. ¹H NMR (500 MHz, CDCl₃) δ 2.48 (dd, J=5.4, 2.0Hz, 1H), 2.81 (t, J=4.4 Hz, 1H), 3.29-3.34 (m, 3H), 5.05-5.08 (m, 2H),6.70 (dd, J=10.2, 2.2 Hz, 1H), 6.73 (t, J=2.2 Hz, 1H), 6.88 (dd, J=8.4,2.4 Hz, 1H), 7.10 (d, J=8.8 Hz, 2H), 7.29-7.46 (m, 6H), 7.89 (d, J=8.8Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 45.9, 45.9, 59.6, 70.2, 107.4,111.8, 112.7, 117.8, 127.5, 128.2, 128.7, 130.5, 130.8, 132.6, 136.4,155.8, 160.3, 162.6.

Compound 4dd=compound 60 (R₁=3-hydroxy, R₂═H). ¹H NMR (500 MHz, CDCl₃) δ2.48 (dd, J=4.5, 1.7 Hz, 1H), 2.82 (1,1H), 3.31 (m, 3H), 6.48 (br.s.,1H), 6.57 (t, J=2.4 Hz, 1H), 6.60 (ddd, J=8.3, 2.1, 0.7 Hz, 1H), 6.71(ddd, J. 8.3, 2.4, 1.0 Hz, 1H), 7.08 (d, J=8.6 Hz, 2H), 7.23 (t, J=7.9Hz, 1H), 7.85 (d, J=9.0 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 46.1, 59.8,108.0, 112.4, 112.6, 118.0, 130.6, 131.0, 132.2, 155.9, 157.8, 162.9.

Compound 5d (R₁=3-benzyloxy, R₂═H). ¹H NMR (500 MHz, CDCl₃) δ 2.18 (dd,J=5.0, 1.4 Hz, 1H), 2.56 (d, J=5.2 Hz, 1H), 3.09 (m, 1H), 3.21 (dd,J=14.3, 7.9 Hz, 1H), 3.55 (dd, J=14.2, 5.6 Hz, 1H), 5.09 (s, 2H),6.69-6.76 (m, 2H), 6.90 (dd, J=8.3, 2.1 Hz, 1H), 7.13 (d, J=8.8 Hz, 2H),7.33-7.38 (m, 2H), 7.40-7.46 (m, 4H), 7.89 (d, J=8.8 Hz, 2H); ¹³C NMR(126 MHz, CDCl₃) δ 24.4, 26.2, 62.7, 70.3, 107.4, 111.9, 112.8, 118.0,127.6, 128.3, 128.8, 130.8, 130.9, 132.1, 136.5, 156.0, 160.4, 162.7.

Compound 5dd=compound 61 (R₁=3-hydroxy, R₂═H). ¹H NMR (500 MHz, CDCl₃) δ2.12 (m, 1H), 2.50 (m, 1H), 3.02 (m, 1H), 3.21 (dd, J=14.1, 7.6 Hz, 1H),3.50 (dd, J=14.1, 5.9 Hz, 1H), 6.60 (m, 2H), 6.73 (d, J=7.9 Hz, 1H),6.86 (br.s., 1H), 7.08 (d, J=9.0 Hz, 2H), 7.22 (t, J=8.6 Hz, 1H), 7.83(d, J=9.0 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 24.3, 26.1, 62.6, 107.9,112.2, 112.6, 118.0, 130.7, 131.0, 131.4, 155.8, 157.8, 162.9.

Compounds of Scheme 14.

Compound 63. Synthesis of compound 63 was done by the similar method forthe preparation of compound 2e using 4-hydroxymethylphenyl-boronic acid(62) and compound 19. ¹H NMR (500 MHz, CDCl₃) δ 2.89 (br.s., 1H),2.94-3.03 (m, 1H), 3.08 (dd, J=13.8, 5.9 Hz, 1H), 3.58-3.66 (m, 2H),3.85-3.91 (m, 1), 4.54 (d, J=17.6 Hz, 1H), 4.61 (s, 1H), 6.78 (t, J=8.6Hz, 1H), 6.91 (t, J=7.9 Hz, 2H), 6.97 (d, J=8.3 Hz, 1H), 7.18 (dd,J=10.0, 8.3 Hz, 1H), 7.31 (t, J=9.0 Hz, 1H), 7.37 (d, J=8.6 Hz, 1H); ¹³CNMR (126 MHz, CDCl₃) δ 39.8, 48.1, 65.1, 69.6, 116.7, 119.5, 119.5,125.5, 128.4, 128.7, 129.0, 129.2, 133.3, 135.7, 157.3 (s).

Conversion from compound 63 to compounds 64-33 was followed by the samemethod as described in Scheme 5.

Compound 3l. ¹H NMR (500 MHz, CDCl₃) δ 2.47 (dd, J=4.8, 2.8 Hz, 1H),2.74 (t, J=4.3 Hz, 1H), 2.80 (br s, 1H), 2.86 (dd, J=13.8, 5.9 Hz, 2H),3.06 (dd, J=13.8, 5.2 Hz, 1H), 3.13 (m, 1H), 4.61 (s, 2H), 6.91 (d,J=8.6 Hz, 2H), 6.97 (d, J=8.6 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 37.9,47.4, 51.2, 64.5, 119.1, 119.2, 128.7, 133.4, 136.6, 156.0, 157.0.

Compound 4l=compound 64. ¹H NMR (500 MHz, CDCl₃) δ 2.43 (dd, J=5.0, 2.2Hz, 1H), 2.76 (dd, J=4.8, 3.8 Hz, 1H), 3.25 (m, 3H), 4.64 (s, 2H), 7.02(dd, J=8.6, 7.9 Hz, 4H), 7.37 (d, J=8.6 Hz, 2H), 7.82 (d, J=9.0 Hz, 2H);¹³C NMR (126 MHz, CDCl₃) δ 45.9, 45.9, 59.5, 64.3, 117.6, 120.5, 128.9,130.5, 132.3, 138.1, 153.9, 162.9.

Compound 5l=compound 33. ¹H NMR (500 MHz, CDCl₃) δ 2.14 (dd, J=5.2, 1.7Hz, 1H), 2.41 (br.s., 1H), 2.52 (d, J=5.2 Hz, 1H), 3.03 (m, 1H), 3.18(dd, J=14.1, 7.6 Hz, 1H), 3.49 (dd, J=14.3, 5.7 Hz, 2H), 4.69 (s, 2H),7.07 (dd, J=8.6, 6.9 Hz, 4H), 7.41 (d, J=8.6 Hz, 2H), 7.84 (d, J=9.0 Hz,2H); ¹³C NMR (126 MHz, CDCl₃) δ 24.3, 26.2, 62.7, 64.5, 117.8, 120.6,129.1, 130.8, 131.9, 138.2, 154.1, 163.0.

Compounds of Scheme 15.

Compound 66=compound 10b. The synthesis of compound 10b was describedpreviously. Conversion from compound 10b to compounds 4m-5m was followedby the same method as described in Scheme 6.

Compound 4m. ¹H NMR (500 MHz, CDCl₃): δ 2.47 (dd, 1H, J=5.0, 2.0 Hz),2.82 (m, 1H), 3.26-3.33 (m, 3H), 3.65 (s, 2H), 3.72 (s, 3H), 7.03-7.05(m, 2H), 7.08-7.10 (m, 2H), 7.32-7.34 (m, 2H), 7.86-7.88 (m, 2H); ¹³CNMR (125 MHz, CDCl₃): δ 40.3, 45.8, 52.1, 59.6, 117.6, 120.6, 130.5,130.9, 131.1, 132.4, 153.8, 162.8, 171.8; HRMS (FAB) calcd for C₁₈H₁₈O₆S(M⁺) 362.0824, found 362.0829.

Compound 5m=compound 67. ¹H NMR (500 MHz, CDCl₃): δ 2.15 (dd, 1H, J=5.5,2.0 Hz), 2.53 (dd, 1H, J=6.5, 2.0 Hz), 3.05 (m, 1H), 3.17 (dd, 1H,J=14.5, 7.0 Hz), 3.51 (dd, 1H, J=14.5, 5.5 Hz), 3.65 (s, 2H), 3.72 (s,3H), 7.03-7.05 (m, 2H), 7.08-7.10 (m, 2H), 7.33-7.34 (m, 2H), 7.85-7.86(m, 2H); ¹³C NMR (125 MHz, CDCl₃): δ 24.2, 26.0, 40.3, 52.1, 62.6,117.7, 120.5, 130.7, 130.9, 131.1, 131.9, 153.8, 162.8, 171.8; HRMS(FAB) calcd for C₁₈H₁₉O₅S₂ (M+H⁺) 379.0674, found 379.0645.

Compound 36a (R₁═R₂═H, n=1). To a stirred solution of 5m (312 mg, 0.83mmol) in toluene (11 mL) was added bis(tributyltin)oxide (1.05 mL, 2.06mmol) at room temperature, and the mixture was stirred at 80° C. for 12h. The solution was cooled to room temperature and was concentrated todryness under reduced pressure. The residue was dissolved inacetonitrile, and the solution was washed with hexane (3×) andconcentrated under reduced pressure to leave the crude tin ester (532mg) as a pale-yellow oil. Subsequently, tin ester was passed through aC₁₈-reverse phase silica gel pad (ODS silica gel 20g, washed with water,1:2 water/acetonitrile and acetonitrile) to afford a mixture of 5m and36a, which was purified by silica gel column chromatography(chloroform/methanol=30/1 to 10/1) to give 36a (195 mg, 65%) as a whitesolid with the recovery of some of 5m (38 mg, 12%). nip 133-134° C.; ¹HNMR (500 MHz, CDCl₃): δ 2.16 (d, 1H, J=4.0 Hz), 2.54 (d, 1H, J-5.5 Hz),3.06 (m, 1H), 3.19 (dd, 1H, J-14.0, 8.0 Hz), 3.52 (dd, 1H, J=14.0, 6.0Hz), 3.68 (s, 2H), 7.05 (br d, 2H, J=8.5 Hz), 7.10 (br d, 2H, J=8.5 Hz),7.35 (brd, 2H, J=8.5 Hz), 7.86 (br d, 2H, J=8.5 Hz); ¹³C NMR (125 MHz,CDCl₃): δ 24.2, 26.0, 40.2, 62.6, 117.8, 120.5, 130.2, 130.7, 131.3,132.0, 154.1, 162.7, 177.1; HRMS (FAB) calcd for C₁₇H₁₇O₅S₂ (M+H⁺)365.0517, found 365.0495; Rf value=0.2 (chloroform/methanol 10/1).

Compounds of Scheme 16.

Compound 69a (R₁═R₂═H). This material was prepared in the same manner asdescribed for 11b in Scheme 7, starting from 68a. ¹H NMR (500 MHz,CDCl₃) δ 2.46 (s, 3H), 4.08 (s, 2H), 7.27-7.33 (m, 5H), 7.38 (d, J=7.0Hz, 2H), 7.43 (d, J=7.6 Hz, 2H).

Conversion from compound 69a to compound 70a was followed by the samemethod as described in Scheme 6.

Compound 70a (R₁═R₂═H). ¹H NMR (500 MHz, CDCl₃) δ 2.16 (dd, J=5.2, 1.8Hz, 1H), 2.54 (dd, J=6.2, 1.6 Hz, 1H), 3.06 (m, 1H), 3.17 (dd, J=14.3,8.1 Hz, 1H), 3.56 (dd, J=14.2, 5.4 Hz, 1H), 4.11 (s, 2H), 7.20 (d, J=7.0Hz, 2H), 7.28 (d, J=7.2 Hz, 1H), 7.35 (t, J=7.5 Hz, 2H), 7.43 (d, J=8.6Hz, 2H), 7.87 (d, J=8.2 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 24.4, 26.2,41.9, 62.6, 126.8, 128.7, 128.9, 129.1, 130.0, 136.4, 139.4, 148.4 (s).

Compound 75a (R—═R₂═H). This material was prepared in the same manner asdescribed for 11b in Scheme 7, starting from 74a. ¹H NMR (300 MHz,CDCl₃) δ 2.42 (s, 3H), 7.39-7.60 (m, 5H), 7.68-7.81 (m, 4H).

Conversion from compound 75a to compound 76a was followed by the samemethod as described in Scheme 6.

Compound 76a (R₁═R₂═H). ¹H NMR (500 MHz, CDCl₃) δ 2.19 (dd, J=5.1, 1.7Hz, 1H), 2.57 (dd, J=6.2, 1.8 Hz, 1H), 3.11 (m, 1H), 3.31 (dd, J=14.4,7.6 Hz, 1H), 3.57 (dd, J=14.4, 6.0 Hz, 1H), 7.54 (t, J=7.8 Hz, 2H), 7.66(t, J=7.4 Hz, 1H), 7.81 (d, J=7.4 Hz, 2H), 7.98 (d, J=8.2 Hz, 2H), 8.08(d, J=8.2 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 24.3, 26.0, 62.6, 128.7,128.9, 130.3, 130.7, 133.7, 136.5, 141.8, 142.9, 195.3.

Compounds of Scheme 17.

Compound 77a (R₁═H). Compound 20a (2.00 g, 12.0 mmol) was dissolved inCH₂Cl₂ (10 mL) in ice-water bath. m-CPBA (13.0 g, 58.0 mmol, 77%) wasadded to the reaction mixture and was stirred at room temperature for 3days. m-Chlorobenzoic acid was filtered and washed with 10% sodiumthiosulfate, and brine. The organic layer was dried under anhydrousMgSO₄, filtered, and concentrated under reduced pressure. The residuewas purified by column chromatography to give the desired product as awhite solid (1.80 g, 70%).

Compound 78a (R₁═H). ¹H NMR (500 MHz, CDCl₃) δ 2.05 (dd, J 5.2, 2.1 Hz,1H), 2.45 (dd, J=6.2, 2.1 Hz, 1H), 2.94-3.01 (m, 1H), 3.11 (dd, J=14.1,7.9 Hz, 1H), 3.47 (dd, J=14.1, 5.5 Hz, 1H), 6.91 (d, J=9.0 Hz, 2H), 7.69(d, J=8.6 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 24.2, 26.2, 62.7, 116.3,128.2, 130.7, 162.6.

Compound 79a (R₁═H, R₂=benzyl). ¹H NMR (500 MHz, CDCl₃) δ 2.12 (dd,J=5.2, 1.6 Hz, 1H), 2.50 (dd, J=6.2, 1.6 Hz, 1H), 3.01-3.08 (m, 1H),3.15 (dd, J=14.2, 8.0 Hz, 1H), 3.51 (dd, J=14.2, 5.4 Hz, 1H), 5.16 (s,2H), 7.11 (d, J=8.8 Hz, 2H), 7.36-7.45 (m, 5H), 7.85 (d, J=8.8 Hz, 2H);¹³C NMR (126 MHz, CDCl₃) δ 24.4, 26.3, 62.7, 70.5, 115.6, 127.6, 128.6,128.9, 130.3, 130.7, 135.7, 163.3.

Compounds of Scheme 18.

Compound 84a (R₁═R₂═H). To a solution of Mg (0.33 g, 13.6 mmol) inanhydrous THE (4 mL), biphenylbromide (3.25 g, 12.8 mmol) in THF (6 mL)was added and the resulting solution was refluxed for 0.5 hours. Thenthe reaction mixture was diluted with THF (10 mL) and cooled down to−78° C. CuBr DMS (0.26 g, 1.29 mmol) was added and stirred for 20minutes, followed by the addition of epichlorohydrin (1.6 mL, 19.2mmol). The reaction mixture was stirred for 1 hour while the reactiontemperature was slowly increased to room temperature. The reaction wasquenched by addition of sat'd NH₄Cl solution and extracted with EtOAc.The combined organic layers were washed with H₂O, 5% HCl, and brine.After removal of solvent, the residue was used for the next reactionwithout further purification. A suspension of crude 83a (3.55 g, 14.4mmol) and K₂CO₃ (5.98 g, 43.3 mmol) in 1:1 solution of MeOH: THF (60 mL)was stirred for 1 hour and then was diluted with diethylether. Thesuspension was filtered and the filtrate was concentrated and theresidue was purified by column chromatography to afford pure 84a (2.37g, 88%).

Compound 85a (R₁═R₂═H). Vinylmagnesium bromide (19.0 mL, 1.0 M in THF)in THF (5 mL) was added CuBr.DMS (0.11 g, 0.55 mmol) at −78° C. After 20minutes, compound 84a (1.15 g, 5.48 mmol) was added to the reactionmixture and stirred for additional 1 hour at −78° C. Stirring wascontinued for 3 hours, while the reaction was warmed to 0° C. Afterquenching the reaction with addition of sat'd NH₄Cl at 0° C. and thesolution was stirred for 0.5 hours and extracted with EtOAc. Afterwashing with water and brine, organic layer was concentrated andpurified by column chromatography (1.06 g, 82%).

Compound 86a (R₁═R₂═H). A solution of compound 85a (1.00 g, 4.20 mmol)and Et₃N (0.73 mL, 5.27 mmol) was added methanesulfonyl chloride (0.39mL, 5.04 mmol) and was stirred for 1 hour. Water was added to thereaction mixture, which was subsequently extracted with EtOAc. Theorganic layer was washed with sat'd NaHCO₃, 1 M KHSO₄, water and brineand evaporated to dryness. The crude material was used for the nextreaction without further purification. A solution of crude material andpotassium thioacetate (2.49 g, 21.8 mmol) in DMF (40 mL) was stirred at50° C. for 17 hours. The reaction mixture was then diluted withether/water and layers were separated. Combined organic layers werewashed with NaHCO₃, H₂O and brine and dried. The residue was purified bycolumn chromatography to give the desired product (0.79 g, 64%) as asemi solid Compound 87a (R₁═R₂═H, R═Bn). t-BuOK (0.79 g, 7.04 mmol) wasadded to a solution of compound 86a (0.52 g, 1.76 mmol) in 1:2 solutionof MeOH:THF (10 mL) and the resulting suspension was stirred for 15minutes, followed by addition of benzyl chloride (1.62 mL, 14.1 mmol).After 3 hours, the reaction was quenched with ammonium hydroxide and wasextracted with diethyl ether. The organic layer was washed with water,5% RCl, and brine. The crude product was purified by columnchromatography to give the desired product (0.55 g, 91%) as a colorlessoil.

Compound 88a (R₁═R₂═H, R═Bn). This compound was prepared in the samemanner as described in Scheme 5.

Compounds of Scheme 19.

Conversion from compound 90a to compound 96a was followed by the samemethod as described in Scheme 14.

Compound 91a (R₁═R₂═H).

Compound 92a (R₁═R₂═H).

Compound 93a (R₁═R₂═H).

Compound 94a (R₁═R₂═H).

Compound 95a (R₁═R₂═H, R₃=Bn).

Compound 96a (R, —R₂═H, R₃=Bn).

Compounds of Scheme 20.

2-Amino-3-bromo-5-nitrophenol (98). A solution of 2-amino-5-nitrophenol(97, 24 g, 156 mmol) in CH₃CN (1 L) was treated with NBS (28.8 g, 160.8mmol) at room temperature. After stirring for 1 hour, the solvent wasremoved to afford brown precipitate, which was taken up with ethylacetate:hexane (1:1). The precipitate was filtered and was used for thenext step without further purification (33 g, 91%). A small amount ofsample was purified by column chromatography on silica gel for analysis;¹H NMR (500 MHz, CD₃OD): δ 4.93 (brs, 3H), 7.50 (d, 1H, J=2.4 Hz), 7.89(d, 1H, J=2.4 Hz); ¹³C NMR (125 MHz, CD₃OD): δ 105.5, 108.7, 121.8,138.5, 143.7, 144.8; HRMS (FAB) calcd for C₆H₅BrN₂O₃ (M⁺) 231.9484,found 231.9479.

3-Bromo-5-nitrophenol (99). Compound 98 (33.0 g, 0.14 mol) was treatedwith sulfuric acid (13.7 mL) and was refluxed in EtOH (550 mL) for 0.5hours, followed by addition of NaNO₂ (23.8 g, 0.35 mol). The resultingmixture was refluxed for additional 1 hour and the volatile wasevaporated. The residue was taken up with ethyl acetate and water. Thelayers were separated and the organic layer was washed with water,saturated sodium bicarbonate, and brine. After removal of solvent, theresidue was purified by column chromatography on silica gel to affordthe title compound (26 g, 85%); ¹H NMR (500 MHz, CD₃OD): δ 5.00 (brs,1H), 7.58 (dd, 1H, J=1.7, 2.3 Hz), 7.47 (t, 1H, J=2.1 Hz), 7.69 (t, 1H,J=1.9 Hz); ¹³C NMR (125 MHz, CD₃OD): δ 110.5, 118.1, 123.7, 125.6,151.0, 160.5; HRMS (FAB) calcd for C₆H₄BrNO₃ (M⁺) 216.9375, found216.9374.

Compound 100a (R₁=iPr). A mixture of compound 99 (4.36 g, 20.0 mmol),K₂CO₃ (5.5 g, 40.0 mmol), and 2-iodopropane (4.0 mL, 40.0 mmol) in DMF(20 mL) was stirred at room temperature overnight. The solution wasdiluted with ethyl acetate and washed with water and brine, dried overMgSO₄, and the volatile was removed under reduced pressure. The residuewas purified by column chromatography to yield the desired product (4.7g, 90%); ¹H NMR (500 MHz, CDCl₃): δ 1.38 (d, 6H, J=6.0 Hz), 4.62(septet, 1H, J=6.0 Hz), 7.33 (t, 1H, J=2.0 Hz), 7.64 (t, 1H, J=2.1 Hz),7.91 (t, 1H, J=1.8 Hz); ¹³C NMR (125 MHz, CDCl₃): δ 21.9, 71.7, 109.3,118.7, 123.1, 125.4, 149.7, 159.2; HRMS (FAB) calcd for C₉H₁₀BrNO₃ (M⁺)258.9844, found 258.9829.

Compound 101a (R₁=iPr, R₂=4-Cl). Compound 100a (4.7 g, 18.1 mmol)dissolved in toluene (45 mL) was treated with sodium carbonate (18 mL, 2M solution) and ethanol (12 mL). 4-Chlorophenylboronic acid (3.06 g,20.1 mmol) was then added to the mixture followed bytetrakis(triphenylphosphine)palladium(0) (0.6 g, 0.54 mmol). Theresulting mixture was refluxed for 3 hours. Brine was added (60 mL) andthe mixture was extracted with CH₂Cl₂. The volatile was evaporated underreduced pressure and the residue was purified by column chromatographyto afford the title compound (4.1 g, 77%); ¹H NMR (500 MHz, CDCl₃): δ1.41 (d, 6H, J=6.0 Hz), 4.70 (septet, 1H, J=6.0 Hz), 7.37 (dd, 1H,J=1.6, 2.4 Hz), 7.47 (d, 2H, J=8.8 Hz), 7.53 (d, 2H, J=8.8 Hz), 7.69 (t,1H, J=2.1 Hz), 7.97 (t, 1H, J=1.7 Hz); ¹³C NMR (125 MHz, CDCl₃): δ 22.0,71.2, 108.5, 114.1, 121.3, 128.5, 129.4, 134.9, 137.3, 142.4, 149.8,159.0; HRMS (FAB) calcd for C₁₅H₁₄ClNO₃ (M⁺) 291.0662, found 291.0688.

Compound 102a (R₁=iPr, R₂═H). Compound 101a (4.0 g, 13.7 mmol) dissolvedin ethyl acetate:ethanol (1:1, mL) was stirred for 4 hours in thepresence of Pd/C under hydrogen atmosphere. The reaction mixture wasfiltered through a small layer of Celite and washed with THF andethanol. The combined filtrate was concentrated under reduced pressureand the residue was used for the next reaction without furtherpurification (3.1 g, quantitative); ¹H NMR (500 MHz, CDCl₃): δ 1.35 (d,6H, J=6.0 Hz), 4.62 (septet, 1H, J=6.0 Hz), 7.10 (dt, 2H, J=1.9, 10.9Hz), 7.32 (t, 1H, J=1.5 Hz), 7.35-7.43 (m, 4H), 7.55 (dd, 2H, J=1.5, 8.4Hz), 10.64 (brs, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 22.1, 70.8, 109.3,114.0, 115.7, 127.4, 128.4, 129.1, 131.2, 139.6, 144.7, 159.5; HRMS(FAB) calcd for C₁₅H₁₇NO (MH⁺) 228.1388, found 228.1372.

Compound 103a (R₁=iPr, R₂=4-Cl). ¹H NMR (500 MHz, CDCl₃) δ 1.38 (d,J=5.9 Hz, 6H), 2.45 (s, 3H), 4.61 (m, 1H), 6.96 (s, 1H), 7.11 (s, 1H),7.17 (s, 1H), 7.40 (d, J=8.4 Hz, 2H), 7.50 (d, J=8.4 Hz, 2H); ¹³C NMR(126 MHz, CDCl₃) δ 22.2, 30.4, 70.5, 116.4, 120.1, 125.1, 128.6, 129.1,129.5, 134.0, 138.7, 142.3, 158.7, 194.0.

Compound 104a (R₁=iPr, R₂═H). ¹H NMR (500 MHz, CDCl₃) δ 1.37 (d, J=5.9Hz, 6H), 2.17 (dd, J=4.9, 1.5 Hz, 1H), 2.53 (d, J=5.9 Hz, 1H), 3.06 (m,1H), 3.23 (dd, J=14.3, 7.4 Hz, 1H), 3.55 (dd, J=14.3, 5.9 Hz, 1H), 4.67(m, 1H), 7.32 (s, 1H), 7.36 (s, 1H), 7.41 (d, J=8.4 Hz, 2H), 7.51 (d,J=8.4 Hz, 2H), 7.63 (s, 1H); ¹³C NMR (126 MHz, CDCl₃) δ 22.0, 24.3,26.1, 62.5, 71.1, 113.3, 118.8, 120.8, 128.5, 129.3, 134.8, 137.5,140.4, 143.0, 159.0.

Compounds of Scheme 21.

Compound 105a (R₁═R₂═H). Compound 14a (2.45 g, 13.2 mmol) was dissolvedin THF (10 mL) and treated with triethylamine (2.2 mL, 15.8 mmol).Allylsulfonyl chloride (2.2 g, 15.8 mmol) in THF (5 mL) was slowly addeddropwise to the above solution at ice-water temperature over 0.5 hours.The resulting mixture was stirred for 1 hour, while the temperature wasgradually warmed to room temperature. Additional triethylamine (2.2 mL,15.8 mmol) was added to the reaction mixture and the resultant solutionwas stirred for 0.5 h. The reaction mixture was filtered through a smalllayer of silica gel and the volatile was evaporated. The residue wastaken up in ethyl acetate and washed with water, 5% NaHCO₃, and brine.The organic layer was dried over MgSO₄, and evaporated. The residue waspurified by column chromatography on silica gel to afford the desiredproduct (2.73 g, 75%); ¹H NMR (500 MHz, CDCl₃) δ 3.87 (d, J=7.4 Hz, 2H),5.32 (d, J=17.3 Hz, 1H), 5.43 (d, J=10.4 Hz, 1H), 5.88 (m, 1H), 6.78(dd, J=8.4, 2.0 Hz, 1H), 6.97 (s, 1H), 6.99-7.07 (m, 2H), 7.16 (t, J=7.4Hz, 1H), 7.28 (t, J=8.2 Hz, 1H), 7.37 (t, J=7.9 Hz, 2H), 7.48 (br.s.,1H); ¹³C NMR (126 MHz, CDCl₃) δ 55.7, 110.8, 114.8, 114.9, 119.3, 123.9,125.0, 130.0, 130.7, 138.5, 156.5, 158.5.

Conversion from compound 105a to compounds 106a-107a was followed by thesame method as described in Scheme 6.

Compound 106a (R₁═R₂═R₃═H). ¹H NMR (500 MHz, CDCl₃) δ 3.24 (dd, J=14.8,3.0 Hz, 1H), 3.33 (dd, J=14.8, 8.9 Hz, 1H), 3.52 (dd, J=11.9, 6.4 Hz,1H), 3.63 (dd, J=12.4, 4.0 Hz, 1H), 4.32 (m, 1H), 6.71 (m, 1H),6.98-7.01 (m, 4H), 7.11 (t, J=7.4 Hz, 1H), 7.20 (dd, J=8.4 Hz, 1H), 7.32(t, J=8.4, 7.4 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 50.4, 53.4, 65.3,67.7, 111.5, 114.9, 115.6, 119.3, 123.9, 129.9, 130.7, 138.3, 156.4,158.3.

Compound 106b (R═R₂═H, R₃=Me). ¹H NMR (500 MHz, CDCl₃) δ 2.4 (s, 3H),3.1-3.3 (m, 2H), 3.9 (d, J=4.0 Hz, 1H), 4.0 (d, J=4.9 Hz, 2H), 4.5(br.s., 1H), 6.8 (d, J=8.9 Hz, 1H), 6.9-7.0 (m, 1H), 7.0 (d, J=7.9 Hz,1H), 7.1 (t, J=7.4 Hz, 1H), 7.2 (t, J=8.4 Hz, 1H), 7.5 (s, 1H), 7.8 (d,J=8.4 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃) δ 21.8, 52.7, 65.4, 71.8, 111.9,115.3, 115.9, 119.4, 123.9, 128.1, 130.0, 130.2, 130.7, 131.9, 138.0,145.6, 156.5, 158.4.

Compound 107a (R₁═R₂═R₃═H). 1H NMR (500 MHz, CDCl₃) δ 2.32 (d, J=5.4 Hz,1H), 2.64 (d, J=5.9 Hz, 1H), 3.19 (m, 1H), 3.26 (dd, J=14.3, 7.4 Hz,1H), 3.57 (dd, J=14.1, 5.7 Hz, 1H), 6.77-6.83 (m, 2H), 6.91 (s, 1H),6.97 (d, J=8.4 Hz, 1H), 7.05 (d, J=8.4 Hz, 2H), 7.18 (t, J=7.4 Hz, 1H),7.28-7.32 (m, 1H), 7.39 (t, J=7.7 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃) δ24.8, 26.7, 57.9, 110.5, 114.6, 115.1, 119.6, 124.2, 130.1, 131.0,137.9, 156.4, 158.9.

Compounds of Scheme 23.

Synthesis of compounds 111a-118a was followed by the same method 110 asdescribed in Schemes 21 and 22.

Compound 111a (R₁=iPr, R₂═H). 1H NMR (500 MHz, CDCl₃) δ 31.41 (d, J=5.9Hz, 6H), 3.92 (d, J=7.9 Hz, 2H), 4.65 (m, 1H), 5.37 (d, J=17.3 Hz, 1H),5.48 (d, J=10.4 Hz, 1H), 5.95 (m, 1H), 6.88 (m, 1H), 6.97 (s, 1H), 7.04(m, 1H), 7.41 (d, J=7.4 Hz, 1H), 7.47 (t, J=7.4 Hz, 2H), 7.59 (d, J=6.9Hz, 2H); ¹³C NMR (125 MHz, CDCl₃): δ 22.2, 55.6, 70.4, 106.8, 111.5,111.7, 125.1, 125.2, 127.3, 128.0, 129.0, 138.4, 140.4, 143.9, 159.3;HRMS (FAB) calcd for C is H₂₁NO₃S (MH⁺) 332.1320, found 332.1313.

Compound 112a (R₁=iPr, R₂═H, R₃=Me). Compound 111a (3.0 g, 9.1 mmol) wastreated with sodium hydride (0.54 g, 13.6 mmol, 60%) in DMF (20 mL) inice-water bath. The resulting suspension was stirred for 1 μl while thetemperature was gradually warmed to room temperature. Iodomethane (1.7mL, 27.2 mmol) was added to above mixture and the resultant solution wasstirred at room temperature overnight. After dilution with ethylacetate, the organics was washed with water, 1 N HCl, and brine and wasdried over MgSO₄. After removal of solvent, the residue was purified byshort-path column chromatography on silica gel to yield the desiredcompound (2.9 g, 91%); ¹H NMR (500 MHz, CDCl₃): δ 1.41 (d, 6H, J=6.0Hz), 3.20 (s, 3H), 3.80 (d, 2H, J=7.9 Hz), 4.61 (septet, 1H, J=5.9 Hz),5.41 (m, 2H), 5.95 (m, 1H), 6.92 (s, 1H), 7.00 (m, 1H), 7.18 (m, 1H),7.40-7.60 (m, 6H); ¹³C NMR (125 MHz, CDCl₃): δ 22.1, 39.2, 54.8, 70.6,106.8, 113.1, 113.6, 117.1, 117.2, 123.7, 127.2, 127.9, 128.5, 128.9,129.1, 139.0, 140.6, 142.2, 159.0; HRMS (FAB) calcd for C₁₉H₂₄NO₃S (MH⁺)346.1477, found 346.1480.

Compound 113a (R₁=iPr, R₂═H, R₃═H). ¹H NMR (500 MHz, CDCl₃) δ 1.32 (d,J=5.9 Hz, 6H), 3.20 (dd, J=14.3, 2.5 Hz, 1H), 3.33 (dd, J=14.6, 9.2 Hz,1H), 3.50 (dd, J=11.6, 6.2 Hz, 1H), 3.62 (dd, J=11.6, 3.2 Hz, 1H), 4.34(m, J=3.0 Hz, 1H), 4.57 (m, 1H), 6.85 (m, 1H), 6.91 (m, 1H), 7.04 (m,1H), 7.30 (t, J=8.4, 7.4 Hz, 1H), 7.37 (t, J=7.4 Hz, 2H), 7.52 (d, J=7.9Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 22.1, 53.4, 58.6, 65.5, 67.9, 70.5,107.9, 112.0, 112.4, 127.3, 128.0, 129.0, 138.3, 140.4, 143.9, 159.2.

Compound 113b (R₁=iPr, R₂═H, R₃=Me). ¹H NMR (300 MHz, CDCl₃): δ 1.35 (d,6H, J=6.0 Hz), 3.15-3.25 (m, 2H), 3.31, 3.34 (2s, 3H), 3.51-3.68 (m,2H), 3.83 (brs, 1H), 4.61 (septet, 1H, J=6.0 Hz), 6.92 (m, 1H), 7.01(dt, 1H, J=1.8, 15.6), 7.14 (dt, 1H, J=1.6, 9.8), 7.35-7.56 (m, 5H); ¹³CNMR (75 MHz, CDCl₃): δ 22.1, 22.2, 38.7, 52.8, 65.5, 67.5, 70.7, 113.5,113.6, 114.1, 114.2, 117.4, 117.5, 127.3, 128.0, 128.6, 129.0, 129.1,138.9, 140.4, 142.4, 159.1; HRMS (FAB) calcd for C₁₉H₂₅NO₅S (M⁺)379.1453, found 379.1462.

Compound 114a (R₁=iPr, R₂═H, R₃═H). ¹H NMR (500 MHz, CDCl₃) δ 1.38 (d,J=5.9 Hz, 6H), 2.32 (dd, J=3.5, 1.5 Hz, 1H), 2.63 (dd, J=4.9, 1.0 Hz,1H), 3.21 (m, 1H), 3.28 (dd, J=14.3, 7.4 Hz, 1H), 3.61 (dd, J=14.1, 5.7Hz, 1H), 4.62 (m, 1H), 6.84 (s, 1H), 6.94 (s, 1H), 7.01 (s, 1H), 7.08(s, 1H), 7.39 (t, J=7.4 Hz, 1H), 7.45 (t, J=7.4 Hz, 2H), 7.56 (d, J=7.4Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 22.2, 24.8, 26.7, 57.7, 70.5, 106.8,111.4, 111.9, 127.3, 128.1, 129.0, 138.0, 140.4, 144.2, 159.4.

Compound 114b (R₁=iPr, R₂=1H, R₃=Me). ¹H NMR (500 MHz, CDCl₃) δ 1.39(dd, J=5.9, 1.5 Hz, 6H), 2.40 (m, 1H), 2.67 (d, J=5.9 Hz, 1H), 3.05 (dt,J=13.9, 8.4 Hz, 1H), 3.19 (m, 1H), 3.41 (d, J=2.5 Hz, 3H), 3.57 (td,J=13.1, 5.4 Hz, 1H), 6.94 (t, J=2.5 Hz, 1H), 6.99 (t, J=2.0 Hz, 1H),7.04 (t, J=2.0 Hz, 1H), 7.13 (t, J=2.0 Hz, 1H), 7.16 (t, J=1.5 Hz, 1H),7.40-7.49 (m, 4H), 7.56 (d, J=6.9 Hz, 2H); ¹³C NMR (125 MHz, CDCl₃): δ22.2, 25.3, 26.7, 38.9, 56.1, 70.5, 113.0, 113.7, 113.9, 117.0, 117.1,127.4, 128.1, 128.6, 129.1, 129.2, 142.5, 142.7, 143.7, 159.0; HRMS(FAB) calcd for C₁₉H₂₄NO₃S₂ (MH⁺) 378.1198, found 378.1200.

Compound 116a (R₁=iPr, R₂=4-Cl, R₃=Me). ¹H NMR (500 MHz, CDCl₃) δ 1.31(d, J=5.9 Hz, 6H), 2.67 (dd, J=20.8, 7.4 Hz, 2H), 3.17 (t, J=7.2 Hz,3H), 3.68 (d, J=10.9 Hz, 3H), 4.51-4.58 (m, 1H), 5.03-5.13 (m, 2H),5.67-5.78 (m, 1H), 6.75 (s, 1H), 6.77 (s, 1H), 6.95 (s, 1H), 7.31 (d,J=8.4 Hz, 2H), 7.42 (d, J=8.4 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 21.9,30.9, 31.9, 36.3 (d, J=3.6 Hz), 50.6 (d, J=7.1 Hz), 69.9, 108.9 (d,J=3.6 Hz), 109.6, 112.9 (d, J=3.6 Hz), 120.1, 120.3, 127.0, 127.1,128.3, 128.7, 129.6, 131.9, 133.4, 139.4, 141.7, 145.8 (d, J=5.3 Hz),158.7.

Compound 117a (R₁=iPr, R₂=4-Cl, R₃=Me). ¹H NMR (500 MHz, CDCl₃) δ 1.35(d, J=5.9 Hz, 6H), 3.18 (dd, J=17.3, 7.9 Hz, 3H), 3.44 (dd, J=11.4, 5.9Hz, 1H), 3.53-3.63 (m, 1H), 3.70 (d, J=11.4 Hz, 3H), 4.01 (s, 1H),4.08-4.15 (m, 1H), 4.54-4.62 (m, 1H), 6.76-6.81 (m, 2H), 6.96 (s, 1H),7.37 (d, J=8.4 Hz, 2H), 7.46 (d, J=8.4 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃)δ 22.1, 30.1, 36.2 (d, J=3.6 Hz), 53.6, 66.8 (d, J=16.0 Hz), 67.0 (d,J=16.0 Hz), 67.2 (d, J=3.6 Hz), 70.2 (d, J=1.8 Hz), 109.3 (d, J=3.6 Hz),109.7 (d, J=3.6 Hz), 110.0, 110.1, 110.8, 113.1 (d, J=3.6 Hz), 113.7 (d,J=2.7 Hz), 128.5, 129.0 (d, J=1.8 Hz), 129.9, 133.8 (d, J=5.3 Hz), 139.4(d, J=3.6 Hz), 142.1 (d, J=14.3 Hz), 145.6 (t, J=4.9 Hz), 159.0 (d,J=8.0 Hz).

Compound 118a (R₁=iPr, R₂=4-Cl, R₃=Me). ¹H NMR (500 MHz, CDCl₃) δ 1.35(d, J=5.9 Hz, 6H), 1.94 (m, 0.5H), 2.24 (td, J=15.7, 6.2 Hz, 0.25H),2.37 (td, J=15.3, 5.4 Hz, 0.25H), 2.46 (m, 0.5H), 2.52 (m, 0.5H), 2.78(m, 1H), 3.18 (m, 1H), 3.23 (d, J=7.9 Hz, 3H), 3.77 (dd, J=11.4, 7.9 Hz,3H), 4.59 (m, 1H), 6.78-6.86 (m, 1H), 6.95-7.03 (m, 1H), 7.34-7.41 (m,J=8.4 Hz, 1H), 7.42-7.48 (m, J=7.7, 7.7 Hz, 3H), 7.54 (t, J=7.4 Hz, 1H),7.66 (dd, J=11.9, 6.9 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃) δ 22.1, 29.5 (d,J=4.5 Hz), 30.5 (d, J=4.5 Hz), 36.1 (d, J=4.5 Hz), 36.4 (d, J=4.5 Hz),46.8, 47.3 (d, J=6.2 Hz), 47.5 (d, J=8.0 Hz), 50.7 (d, J=7.1 Hz), 50.8(d, J=6.2 Hz), 70.2 (d, J=1.8 Hz), 109.2 (d, J=3.6 Hz), 109.9 (d, J=3.6Hz), 110.0, 110.4, 113.2 (d, J=3.6 Hz), 113.9 (d, J=3.6 Hz), 127.3,128.5, 128.6, 128.7, 129.0 (d, J=2.7 Hz), 132.1 (t, J=2.7 Hz), 132.1,132.2, 133.8 (d, J=6.2 Hz), 139.5 (d, J=2.7 Hz), 142.1, 142.2, 145.6(dd, J=8.0, 4.5 Hz), 159.0 (d, J=6.2 Hz).

Compounds of Scheme 24.

Compound 121a (R₁=Bn, R₂═R₃═H). Biphenylsulfonyl chloride (1.59 g, 6.30mmol) was added to a solution of compound 119a (1.00 g, 6.61 mmol) in3:1 mixture of THF:water in ice-water bath. The resulting mixture wasstirred at room temperature overnight. Sodium bicarbonate (0.56 g, 6.94mmol) was added and the resulting solution was stirred for 3 h andsolvent was concentrated. The resultant was diluted with EtOAc and waterand then layers were separated. The organic layer was washed with 5% HCland water and was concentrated to dryness. The crude product waspurified by column chromatography to give the desired product (1.97 g,81%).

Compound 122a (R₁=Bn, R₂═R₃═H). Methanesulfonyl chloride (0.58 mL, 7.42mmol) was added to a solution of compound 121a (1.82 g, 4.95 mmol) andEt₃N (1.72 mL, 12.4 mmol) in acetonitrile (30 mL) in ice-water bath andstirred for 10 min. After stirring at room temperature for additional 10min, the reaction mixture was diluted with 200 mL of MeOH and cooleddown to ice-water bath. Potassium carbonate (1.37 g/10 mL water) wasadded to the reaction mixture and stirred for 3 h at room temperature.Organic solvents were removed and then diluted with EtOAc and water. Thelayers were separated and the organic layer was washed with water andbrine and concentrated. The crude product was purified by columnchromatography to give the desired product (1.27 g, 73%).

Compound 123a (R₁=Bn, R₂═R₃═H, n=1). Vinyl magnesium bromide (4.5 mL,1.0 M in THF) was added to a solution of CuI (85 mg, 0.45 mmol) in THF(3 mL) at −78° C. After 20 minutes, compound 122a (313 mg, 0.90 mmol)was added to the reaction mixture. The reaction mixture was slowlywarmed to room temperature over 1.5 hours. After quenching with 5% HCl,the reaction mixture was extracted with EtOAc and organic layer waswashed with 5% HCl and brine and volume was reduced. The crude productwas purified by column chromatography to give the desired product (237mg, 70%) as a yellow oil.

Compound 123b (R₁=Bn, R₂═R₃═H, n=2). This material was prepared in thesame manner as described for 123a, with the exception thatallylmagnesium bromide was used in place of vinylmagnesium bromide.

Conversion from compound 123 to compound 124, 125 was followed by thesame method as described in Scheme 5.

Compound 124a (R₁=Bn, R₂═R₃═H, n=1).

Compound 124b (R₁=Bn, R₂═R₃═H, n=2).

Compound 125a (R₁=Bn, R₂═R₃═H, n=1).

Compound 125b (R₁=Bn, R₂═R₃═H, n=2).

Compounds of Scheme 25.

Compound 130a (R₁=Bn, R₂═R₃—H). A solution of compound 129a (183 mg,1.25 mmol), 107a (330 mg, 1.31 mmol), and Bt₃N (0.21 mL, 1.50 mmol) inCH₂Cl₂ (6 mL) was stirred for 1 h at room temperature. The reactionmixture was washed with water and organic solvent was concentrated. Thecrude product was purified by column chromatography to give the desiredproduct (389 mg, 86%) as a white solid.

Conversion from compound 130a to compound 131a was followed by the samemethod as described in Scheme 5.

Compound 131a (R₁=Bn, R₂═R₃═H).

Compounds of Scheme 26.

Compound 133a (R₁=Bn, R₂═R₃═H). Compound 132 (1.17 mL, 11.9 mmol) wasadded to a solution of phenoxyphenylmagnesium bromide (3.30 g, 12.0mmol) in anhydrous THF (20 mL) in ice-water bath. After 15 minutes,stirring was continued at room temperature for 1 h and reaction wasquenched with sat'd NH₄Cl solution. After extraction with EtOAc, theorganic layer was washed with water and brine. The crude product waspurified by column chromatography to give the desired product (2.39 g,79%) as a white solid.

Compound 134a (R₁=Bn, R₂═R₃═H). A mixture of compound 133a (100 mg, 0.39mmol), TPAP (14 mg, 0.039 mmol), and NMO (138 mg, 1.30 mmol) in CH₂Cl₂(mL) was stirred for 5 minutes and concentrated to dryness. The crudeproduct was purified by column chromatography to give the desiredproduct (88 mg, 89%) as a white solid.

Compound 135a (R₁=Bn, R₂═R₃═H). A mixture of compound 134a (88 mg, 0.35mmol) and NBS (68 mg, 0.39 mmol) in 3:1 solution of THF:water wasstirred at room temperature for 1 hour. 2.5 N NaOH (280 μL) was added tothe reaction mixture and stirred for 3 h. The reaction mixture wasdiluted with diethyl ether and washed with brine. The crude product waspurified by column chromatography to give the desired product (75 mg,81%) as a colorless oil.

Conversion from compound 135a to compound 136a was followed by the samemethod as described in Scheme 5.

Compound 136a (R₁=Bn, R₂═R₃═H).

Compound 137a (R₁═Bn, R₂═R₃═H). A mixture of compound 136a (66 mg, 0.23mmol), NH₂OH—HCl (24.2 mg, 0.38 mmol), and NaOAc (28.6 mg, 0.38 mmol) in2:1 solution of EtOH:water was stirred at room temperature for 2 h.Additional batch of NH₂OH—HCl and NaOAc was to the reaction mixture andstirring was continued for 4 h. The resulting solution was diluted withEtOAc and water and layers were separated. Organic layer was washed withsat'd NaHCO₃, water and brine. The crude product was purified by columnchromatography to give the desired product (53 mg, 76%) as a colorlessoil.

Compounds of Scheme 27.

Preparation of compounds 139a-143a was followed by the same method asdescribed in Scheme 22.

Compound 139a (R₁═R₂═H).

Compound 140a (R₁═R₂═H).

Compound 141a (R₁═R₂═H).

Compound 142a (R₁═R₂═H).

Compound 143a (R₁═R₂═H).

Compounds of Scheme 28.

Compound 150a (R¹═H). A solution of compound 149a (1.10 g, 5.12 mmol) inTHF (5 mL) was added dropwise to a stirred suspension of KH (230 mg,5.63 mmol) in THF (10 mL) at room temperature, and the whole was stirredat the same temperature for 5 minutes. A hexane solution oftriethylborane (1.0 M, 6.4 mL, 6.4 mmol) was added to the reactionmixture at room temperature, and the whole was stirred at the sametemperature for 5 minutes. After addition of allylbromide (1.43 mL, 15.4mmol) at room temperature, the reaction mixture was stirred at the sametemperature for 36 hours. After addition of water, the solvent wasremoved under reduced pressure, and the resultant residue waspartitioned with CH₂Cl₂ and water. The aqueous layer was extracted withCH₂Cl₂, and the combined organic layers were washed with water andbrine, and then dried over Na₂SO₄.

After concentration under reduced pressure, the resultant residue waspurified by silica gel column chromatography (ethyl acetate:hexane=1:30)to give the desired product (837 mg, 64%) as a pale yellow solid withrecovery of compound 149a (254 mg, 23%); Compound 150a: ¹H NMR (500 MHz,CDCl₃): δ 1.83-1.88 (m, 2H), 1.93-1.97 (m, 2H), 2.50-2.55 (m, 2H),2.62-2.65 (m, 2H), 2.75-2.78 (m, 2H), 3.12 (t, 2H, J=7.5 Hz), 5.02 (dq,1H, J=10.5, 1.0 Hz), 5.10 (dq, 1H, J=17.0, 2.0 Hz), 5.93 (ddt, 1H,J=17.0, 10.5, 6.5 Hz), 7.43 (d, 1H, J=8.0 Hz), 7.85 (dd, 1H, J=8.0, 1.5Hz), 8.01 (d, 1H, J=1.5 Hz); ¹³C NMR (125 MHz, CDCl₃): δ 8.5, 37.8,110.7, 113.3, 115.2, 118.0, 122.5, 132.2, 133.3, 137.5, 154.0, 158.1,199.0; HRMS (FAB) calcd for C₁₇H₁₉O₂ (M+H⁺) 255.1385, found 255.1402.

Compound 149a: m.p. 66-67° C.; ¹H NMR (300 MHz, CDCl₃): δ 1.82-1.90 (m,2H), 1.92-2.00 (m, 2H), 2.61-2.67 (m, 2H), 2.65 (s, 3H), 2.75-2.80 (m,2H), 7.43 (d, 1H, J=8.4 Hz), 7.84 (dd, 1H, J=1.2, 8.4 Hz), 8.00 (d, 1H,J=1.2 Hz); ¹³C NMR (75 MHz, CDCl₃): δ20.2, 22.4, 22.6, 23.5, 26.6,110.9, 113.3, 117.8, 122.8, 132.4, 133.2, 153.9, 158.1, 197.6; HRMS(FAB) calcd for C₁₄H₁₅O₂ (M+H⁺) 215.1072, found 215.1056.

Compound 151a (R₁═H). NBS (650 mg, 3.66 mmol) was added to a stirredsolution of compound 150a (780 mg, 3.05 mmol) in THF-water (3:1, 15 mL)at room temperature, and the whole was stirred at the same temperaturefor 1 hour in the dark. Aqueous 2.5 M NaOH (3.7 mL, 9.25 mmol) was addedat room temperature, and the whole was stirred at the same temperaturefor 30 minutes. After addition of a few drops of allyl alcohol, themixture was stirred for 10 minutes. After dilution with ether and brine,the aqueous layer was extracted with ether. The combined organic layerswere washed with water and brine, dried over MgSO₄ and concentratedunder reduced pressure.

The resultant residue was purified by silica gel column chromatography(1% triethylamine in ethyl acetate:hexane=1:8) to give the titlecompound (540 mg, 65%) as a white solid; ¹H NMR (500 MHz, CDCl₃): δ1.83-1.90 (m, 3H), 1.93-1.98 (m, 2H), 2.19 (m, 1H), 2.56 (dd, 1H, J=2.5,5.0 Hz), 2.62-2.65 (m, 2H), 2.76-2.81 (m, 3H), 3.08 (m, 1H), 3.20 (t,2H, J=7.5 Hz), 7.44 (d, 1H, J=8.0 Hz), 7.86 (dd, 1H, J=1.0, 8.0 Hz),8.02 (d, 1H, J=1.0 Hz); ¹³C NMR (125 MHz, CDCl₃): δ 4.6, 47.4, 51.7,110.7, 113.4, 118.0, 122.5, 132.0, 133.4, 154.0, 158.3, 198.5; HRMS(FAB) calcd for C₁₇H₁₉O₃ (M+H⁺) 271.1334, found 271.1343.

Compound 152a (R₁═H). Thiourea (349 mg, 4.60 mmol) was added to astirred solution of compound 151a (500 mg, 1.84 mmol) in MeOH (11 mL) atroom temperature, and the whole was stirred at the same temperatureovernight. After concentration under reduced pressure, the resultant wasdissolved in ethyl acetate. The ethyl acetate solution was washed withwater and brine, dried over Na₂SO₄, and concentrated under reducedpressure. The resultant residue was purified by silica gel columnchromatography (ethyl acetate:hexane=1:20) to give the desired product(410 mg, 78%) as a white solid; ¹H NMR (500 MHz, CDCl₃): δ 1.72 (m, 1H),1.84-1.88 (m, 2H), 1.93-1.98 (m, 2H), 2.25 (dd, 1H, J=1.0, 5.5 Hz), 2.52(m, 1H), 2.56 (dd, 1H, J=1.0, 6.5 Hz), 2.62-2.65 (m, 2H), 2.76-2.79 (m,2H), 3.07 (m, 1H), 3.19-3.29 (m, 2H), 7.43 (d, 1H, J=8.5 Hz), 7.86 (dd,1H, J=1.5, 8.5 Hz), 8.02 (d, 1H, J=1.5 Hz); ¹³C NMR (125 MHz, CDCl₃): δ20.9, 22.4, 22.6, 23.6, 26.3, 31.0, 35.7, 38.0, 110.7, 113.4, 118.0,122.5, 132.0, 133.4, 154.0, 158.3, 198.6; HRMS (FAB) calcd for C₁₇H₁₉O₂S(M+H⁺) 287.1106, found 287.1104.

Compound 153a (R¹═H). A solution of hydroxylamine hydrochloride (266 mg,3.82 mmol) and sodium acetate (314 mg, 3.82 mmol) in water (1 mL) wasadded dropwise to a stirred solution of compound 152a (365 mg, 1.27mmol) in EtOH—CH₂Cl₂ (3:1, 12 mL) at room temperature, and the whole wasstirred at the same temperature for 6 h. After dilution with water, themixture was extracted with ethyl acetate. The combined organic layerswere washed with water and brine, dried over Na₂SO₄, and concentratedunder reduced pressure. The resultant residue was purified by silica gelcolumn chromatography (ethyl acetate:hexane=1:10) to give the desiredproduct (320 mg, 83%) as a white solid; ¹H NMR (500 MHz, CDCl₃): δ 1.76(m, 1H), 1.83-1.87 (m, 2H), 1.92-1.97 (m, 2H), 2.14-2.21 (m, 2H), 2.48(d, 1H, J=6.5 Hz), 2.62 (t, 2H, J=6.0 Hz), 2.75 (t, 2H, J=6.0 Hz), 2.96(qn, 1H, J=6.5 Hz), 3.02 (m, 1H), 3.14 (m, 1H), 7.40 (d, 1H, J=7.5 Hz),7.50 (d, 1H, J=7.5 Hz), 7.67 (s, 1H), 9.02 (s, 1H); ¹³C NMR (125 MHz,CDCl₃): δ 20.4, 22.5, 22.8, 23.5, 25.9, 26.0, 33.3, 35.5, 108.8, 113.0,118.3, 120.5, 130.0, 130.4, 154.4, 155.7, 158.9; HRMS (FAB) calcd forC₁₇H₂₀O₂NS (M+H⁺) 302.1215, found 302.1212.

Compounds of Scheme 29.

Compound 155a (R₁=3-benzyloxy). A mixture of 3-benzyloxybenzaldehyde(9.5 g, 44.7 mmol) and ethyl cyanoacetate (5.2 mL, 48.9 mmol) in benzene(50 mL) was refluxed in the presence of catalytic amount of piperidine(0.5 mL) for 4 h. Evaporation of solvent under reduced pressure resultedin precipitate which was purified by recrystallisation from ethanol(11.0 g, 80%); ¹H NMR (400 MHz, CDCl₃): δ 1.42 (t, 3H, J=7.20 Hz), 4.40(q, 1H, J=7.0 Hz), 5.14 (s, 2H), 7.19 (dd, 1H, J=2.4, 8.0 Hz), 7.35-7.67(m, 9H), 8.22 (s, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 14.3, 63.0, 70.3,103.3, 115.7, 121.0, 124.6, 127.8, 128.4, 128.8, 130.5, 132.8, 136.4,155.2, 159.2, 162.6; HRMS (ESI) calcd for C₁₉H₁₇NNaO₃ (M+Na⁺) 330.1106,found 330.1112.

Compound 156a (R₁=3-benzyloxy, R₂═H). Potassium cyanide (2.85 g, 43.8mmol) dissolved in water (5 mL) was added to a solution of compound 155a(7.5 g, 24.4 mmol) in EtOH (40 mL). After reflux for 4 h, the reactionmixture was cooled down and was diluted with 1 N NaOH (25 mL) and 15%NaCl (400 mL), followed by extraction with CH₂Cl₂. The volatile wasevaporated under reduced pressure and the residue was purified by columnchromatography on silica gel to afford the desired product (5.8 g, 91%);¹H NMR (400 MHz, CDCl₃): δ 2.96 (d, 2H, J=6.8 Hz), 4.13 (t, 1H, J=6.8Hz), 5.10 (s, 2H), 7.00-7.04 (m, 3H), 7.36-7.46 (m, 5H); ¹³C NMR (100MHz, CDCl₃): δ 24.8, 34.2, 70.4, 114.2, 115.4, 115.9, 117.9, 119.8,127.8, 128.4, 128.9, 131.1, 133.8, 136.4, 159.7; HRMS (ES) calcd forC₁₇H₁₄N₂NaO (M+Na⁺) 285.1004, found 285.1010.

Compound 157a (R₁=3-benzyloxy, R₂═H). Dibal-H (52.5 mL, 52.5 mmol, 1.0 Nsolution in toluene) was added dropwise to a solution of compound 156a(5.3 g, 20.2 mmol) in benzene (140 mL) at ice-water temperature. Theresulting mixture was stirred for 2 h, while the temperature wasgradually warmed to room temperature over 2 h. Sodium dihydrogenphosphate (350 mL, 1.5 N aqueous solution) was added dropwise to theabove solution and the resulting solution was refluxed for 1 hour. Thereaction mixture was filtered through a layer of Celite and washed withethyl acetate. The layers of the combined filtrate were separated andthe organic layer was washed with water, brine, dried over MgSO₄, andevaporated under reduced pressure. The residue was purified by columnchromatography on silica gel to afford the desired product (2.0 g, 40%);¹H NMR (400 MHz, CDCl₃): δ 5.15 (s, 2H), 7.00-7.04 (m, 3H), 7.36-7.46(m, 5H), 8.27 (brs, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 70.1, 106.7, 111.8,112.2, 115.0, 118.3, 119.1, 124.8, 127.8, 128.1, 128.8, 129.8, 137.3,137.5, 159.3; HRMS (ES) calcd for C₁₇H₁₅NNaO (M+Na⁺) 272.1051, found272.1056.

Compound 158a (R₁=3-benzyloxy, R₂═H, n=1, X═O). Glycidol (2.6 mL, 40.0mmol) dissolved in ethyl acetate (200 mL) was treated with triethylamine(5.6 mL, 40.2 mmol). Diphosgene (4.6 mL, 38.4 mmol) was slowly added tothe above solution at −20° C., and the reaction mixture was stirred atroom temperature for 2 h. The precipitate was filtered off and thefiltrate was evaporated. The residue was purified by short-path columnchromatography on silica gel yielding 2,3-epoxypropyl chloroformate.Pyrrole 144a (2.0 g, 8.0 mmol) in anhydrous acetonitrile (30 mL) wastreated with NaH (0.64 g, 16.0 mmol, 60%) at ice-water temperature. Theresulting mixture was stirred while temperature was gradually warmed toroom temperature over 1 hour. After cooling in ice-bath, 2,3-epoxypropylchloroformate (2.2 g, 16.4 mmol) in acetonitrile (5 mL) was added to thereaction mixture and was stirred while the temperature was graduallywarmed to room temperature over 2 h. After stirring at 50° C. foradditional 1 hour, the resulting mixture was filtered through a smalllayer of silica gel and the filtrate was evaporated. The residue wastaken up in ethyl acetate and was washed with water, brine, and driedover MgSO₄.

After removal of solvent, the residue was purified by columnchromatography on silica gel to afford the desired product (1.5 g, 55%);¹H NMR (400 MHz, CDCl₃): δ 2.73 (dd, 1H, J=2.4, 4.9 Hz), 2.92 (dd, 1H,J=4.0, 4.9 Hz), 3.35 (sextet**, 1H, J=3.2 Hz), 4.19 (dd, 1H, J=6.5, 12.2Hz), 4.71 (dd, 1H, J=3.2, 12.2 Hz), 5.11 (s, 2H), 6.59 (dd, 1H, J=1.6,3.2 Hz), 6.89 (dd, 1H, J=2.8, 8.4 Hz), 7.16-7.49 (m, 9H), 7.58 (m, 1H);¹³C NMR (100 MHz, CDCl₃): δ 44.8, 49.3, 68.1, 70.3, 111.7, 112.6, 113.3,116.3, 118.7, 121.3, 127.8, 128.2, 128.9, 130.1, 135.6, 137.2, 150.3,159.4; HRMS (ESI) calcd for C₂₁H₁₉NNaO₄ (4+Na⁺) 372.1212, found372.1215.

Compound 158b (R₁=3-hydroxy, R₂═H, n=1, X═O). Compound 158a (0.7 g, 2.0mmol) was stirred for 2 hours in the presence of Pd(OH)₂ (0.2 g) inethyl acetate:THF (1:1, 15 mL) under hydrogen atmosphere. The reactionmixture was filtered through a layer of celite and washed with methanol.The filtrate was evaporated under reduced pressure and the residue waspurified by column chromatography on silica gel to afford the desiredproduct (0.35 g, 67%); ¹H NMR (400 MHz, CDCl₃): δ 2.74 (dd, 1H, J=2.8,4.5 Hz), 2.93 (dd, 1H, J=4.1, 4.8 Hz), 3.36 (m, 1H), 4.20 (dd, 1H,J=6.5, 12.2 Hz), 4.72 (dd, 1H, J=2.4, 12.2 Hz), 6.56 (dd, 1H, J=1.6, 4.0Hz), 6.73 (dd, 1H, J=2.4, 8.0 Hz), 7.00-7.26 (m, 4H), 7.33 (m, 1H), 7.55(m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 45.0, 49.5, 68.1, 111.8, 112.7,114.2, 118.1, 121.3, 130.2, 135.6, 150.3, 156.5; HRMS (ESI) calcd forC₁₄H₁₃NNaO₄ M+Na⁺) 282.0742, found 282.0752.

Compound 158c (R₁=3-hydroxy, R₂═H, n=1, X═S). A mixture of compound 158b(300 mg, 1.2 mmol) and thiourea (120 mg, 1.6 mmol) in anhydrous methanol(10 mL) was stirred at room temperature overnight. The volatile wasevaporated under reduced pressure and the residue was purified by columnchromatography on silica gel to afford the desired product (200 mg,63%); ¹H NMR (400 MHz, CDCl₃): δ 2.37 (dd, 1H, J=1.6, 5.7 Hz), 2.61 (d,1H, J=5.7 Hz), 3.26 (quintet, 1H, J=5.7 Hz), 4.40 (dd, 1H, J=7.3, 11.4Hz), 4.48 (dd, 1H, J=6.5, 11.3 Hz), 6.56 (dd, 1H, J=1.6, 3.2 Hz), 6.74(dd, 1H, J=2.4, 8.1 Hz), 7.02 (dd, 1H, J=1.6, 2.4 Hz), 7.12 (d, 1H,J=8.1 Hz), 7.22-7.26 (m, 2H), 7.36-7.46 (m, 5H), 7.33 (dd, 1H, J=1.6,3.3 Hz), 7.55 (m, 1H), 8.27 (brs, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 24.0,30.5, 71.3, 111.7, 112.7, 116.2, 118.4, 121.3, 128.5, 130.2, 135.7,150.2, 156.2; HRMS (ESI) calcd for C₁₄H₁₃NNaO₃S (M+Na⁺) 298.0514, found298.0522.

Compounds 159a-f were prepared in the same manner as described for 158,with the exception that were used epichlorohydrin and3,4-epoxybutylchloride in place of 2,3-epoxypropylchloroformate.

Compound 159a (R₁=3-benzyloxy, R₂═H, n=1, X═O).

Compound 159b (R₁=3-hydroxy, R₂═H, n=1, X═O).

Compound 159c (R₁=3-hydroxy, R₂═H, n=1, X═S).

Compound 159d (R₁=3-benzyloxy, R₂═H, n=2, X═O).

Compound 159e (R₁=3-hydroxy, R₂═H, n=2, X═O).

Compound 159f (R₁=3-hydroxy, R₂-═H, n-2, X═S).

Example 4 Other Compounds of the Invention

FIG. 9 illustrates several other species and classes of compounds of theinvention. These compounds can be prepared according to the methodsdescribed in Example 3, by using appropriate starting materials, and byusing other techniques well know to those of skill in the art.

All publications, patents, and patent documents referred to herein areincorporated by reference, as though individually incorporated byreference. The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it will beapparent to those skilled in the art that many variations andmodifications may be made while remaining within the spirit and scope ofthe invention.

1. A compound of formula I:

wherein R¹ is (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy,aryl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkoxy,heteroaryl(C₁-C₆)alkoxy, aryl, heteroaryl, hydroxy, SR⁵, NR⁵R⁵, orabsent; R² is CH₂, carbonyl, SO₂, or OH; L is CH₂, NR⁵, or O; R³ and R⁴are each independently hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, aryl, heteroaryl, carboxy, cyano,nitro, halo, trifluoromethyl, trifluoromethoxy, SR⁵, SO₂N(R₅)₂, NR⁵R⁵,or CO₂R⁵; each n is independently 0 to 4; each R⁵ is independently H,(C₁-C₆)alkyl, (C₁-C₆)alkanoyl, (C₆-C₁₀)aroyl, aryl, aryl(C₁-C₆)alkyl,heteroaryl, heteroaryl(C₁-C₆)alkyl, or a nitrogen protecting group; X isO; D is S, SO, SO₂, C═N—OH, or carbonyl; E is a direct bond, or(C₁-C₆)alkyl; J is S, O, or NR⁵; G, T, and Q are each independently H,(C₁-C₆)alkyl, or cyano; any alkyl, amino, aryl, heteroaryl, orcycloalkyl is optionally substituted with 1 to about 5 (C₁-C₆)alkyl,(C₁-C₆)alkoxy, aryl, heteroaryl, aryl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl, nitro, halo, amino, or hydroxy groups; or apharmaceutically acceptable salt thereof; provided that when L is CH₂ orO, and R² is CH₂, R¹ is not (C₁-C₆)alkyl; when L is O and R² iscarbonyl, R¹ is not (C₁-C₆)alkyl; and when L is NR⁵, R² is not CH₂. 2.The compound of claim 1 wherein R¹ is (C₁-C₆)alkyl, (C₁-C₆)alkoxy,heteroaryl(C₁-C₆)alkoxy, halo(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, or aryl;and R² is CH₂, carbonyl, or SO₂.
 3. The compound of claim 1 wherein L isCH₂, O, or NR⁵ wherein R⁵ is H, (C₁-C₆)alkyl, or a nitrogen protectinggroup.
 4. The compound of claim 1 wherein R³ is hydroxy, halo, amino,hydroxyphenyl, halophenyl, or SO₂N(R₅)₂ wherein each R⁵ of R³ is H. 5.The compound of claim 1 wherein R⁴ is hydroxy, halo, amino, or SO₂N(R₅)₂wherein each R⁵ of R⁴ is H.
 6. The compound of claim 1 wherein D is SO₂,or carbonyl.
 7. The compound of claim 1 wherein E is a direct bond, CH₂,or (C₁-C₆)alkyl, optionally substituted with 1 to 5 halo groups.
 8. Thecompound of claim 1 wherein J is S, O, or NH.
 9. The compound of claim 1wherein G, T, and Q are each H.
 10. The compound of claim 1 wherein thecompound of formula I is a compound of the formula:


11. The compound of claim 10 wherein J is S or O; the groups G, T, and Qare each H; and each n is
 0. 12. The compound of claim 10 wherein L isCH₂, R² is carbonyl, and R¹ is (C₁-C₆)alkoxy, aryl(C₁-C₆)alkoxy,heteroaryl(C₁-C₆)alkoxy, or hydroxy.
 13. The compound of claim 10wherein L is NR⁵ wherein R⁵ is H, R² is SO₂ or carbonyl, and R¹ is(C₁-C₆)alkyl or (C₁-C₆)alkoxy.
 14. The compound of claim 10 wherein L isO, R² is SO₂ or carbonyl, and R¹ is (C₁-C₆)alkyl or optionallysubstituted aryl.
 15. The compound


16. The compound


17. A pharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable carrier.
 18. A composition comprising acompound of claim 1 and a chemotherapeutic agent.
 19. A method ofinhibiting a matrix metalloproteinase in a cell comprising contacting acell with an amount of a compound of claim 1 that is effective toinhibit a matrix metalloproteinase.
 20. The method of claim 19 whereinthe contacting is in vitro or in vivo.